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PMC4472642

supplementary material 1 ( pptx 117 kb ) supplementary material 2 ( avi 825 kb ) supplementary material 3 ( avi 862 kb ) supplementary material 4 ( avi 532 kb ) supplementary material 5 ( avi 802 kb ) supplementary material 6 ( avi 691 kb ) asif h. khan , ian b. a. menown , alastair graham and john a. purvis declare no conflicts of interest . this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .

we present the case of 56-year - old woman who required complex coronary artery bypass grafting for high - risk anomalous left main coronary artery ( lmca ) originating from right coronary cusp including conventional reimplantation of the lmca plus left internal mammary artery ( lima ) graft to the left anterior descending ( lad ) and saphenous vein graft ( svg ) to the left circumflex ( lcx ) . on subsequent cardiac computed tomography screening and cardiac catheterization , the lima graft was occluded after just a few centimeters , but the svg graft was patent with good run - off into the native lcx and also filled the lad retrogradely . the reimplanted left main stem demonstrated at least moderate ostial stenosis although pressure wire assessment of this was not significant ( fractional flow reserve 0.89 ) , probably due to good retrograde filling of the lad from the svg to lcx , therefore , we did not proceed with ostial lmca stenting . she remains on yearly review with a low threshold for further revascularization should the svg to lcx develop progressive stenosis . this case illustrates how patients with anomalous lmca may sometimes benefit from grafting in addition to conventional reimplantation.electronic supplementary materialthe online version of this article ( doi:10.1007/s40119 - 015 - 0039-x ) contains supplementary material , which is available to authorized users .

Electronic supplementary material Conflict of interest Compliance with ethics guidelines Open Access

PMC2911849

comprehensive , top - level registries of biological resources are currently provided by the nucleic acids research molecular biology database collection , the biomedcentral catalog of databases on the web ( http://databases.biomedcentral.com ) and the bioinformatics.ca links directory . however , they do not collect extensive metadata beyond a brief description of the resource and url , can only be browsed by the category each registry has assigned or searched by the resource name , and lack much of the detailed information that the community requires . a number of projects [ e.g. casimir ( coordination and sustainability of mouse informatics resources ) and enfin ] have identified this problem and are producing database of databases ( registries ) for their field of expertise . a registry of resources needs to be more than just a list of databases and textual descriptions to be useful to the biological and bioinformatics communities . to achieve its aim of helping scientists find the most relevant resource for their needs , it needs to provide at the very least browsing and searching by the type of data contained in each resource , i.e. the biological scope of the resource . a typical approach , as used by all the registries described above and the mrb ( mouse resource browser ) registry developed by a number of the authors of this article , is for a community to define a list of categories ( a controlled vocabulary ) that covers their scientific domain and then to tag each resource with one or more of these terms . use of existing and newly developed ontologies for these tags would certainly facilitate future interoperability of the various registries being developed . while developing mrb , user feedback suggested that it would be helpful if users could go beyond simple categorization of the scope of resources to discover metadata describing database operations and functionality . we therefore set out to capture the utility , accessibility and ease of use of a resource , along with its potential interoperability with other tools and databases . the types of questions that we wanted to be able to answer from this metadata included whether the resource uses automated or manual curation , how often it updates and whether there is a way to track back to different versions , does it provide good technical documentation and user support , does it use recognized standards to record and structure its data , and finally does it go beyond simple web browsing to allow programmatic access and output in standard formats ? data are always easier to capture and search if a consistent standard is used and we therefore developed a database description framework ( ddf ; table 1 ) as part of the casimir project . although produced for the mrb , the ddf is generically applicable to any biological database and can be adapted for the requirements of any biological community . for each heading or category , there is a three - tier assessment criterion , a number chosen for simplicity and ease of use . the aim of the ddf is not to make value judgements about a resource , but to summarise what it does and what functionalities it supports , with the categories simply reflecting the degree of complexity or sophistication of the database . what is useful or relevant for some databases need not be so for others , and each needs to be assessed in terms of its own remit and user community . the ddf is also intended to be helpful in disseminating and supporting good database practice , in providing backing for resources aspiring to improve the levels of their service , and in giving objective criteria that can be used by external assessors to measure a resource s progress towards their stated goals . table 1.the casimir database description framework ( ddf)categorylevel 1level 2level 3quality and consistencyno explicit process for assuring consistencyprocess for assuring consistency , automatic curation onlyprocess for assuring consistency with manual curationcurrencyclosed legacy database or last update more than a year agoupdates or versions more than once a yearupdates or versions more than once a monthaccessibilityaccess via browser onlyaccess via browser and database reports or database dumpsaccess via browser and programmatic access ( well defined api , sql access or web services)output formatshtml or similar to browser onlyhtml or similar to browser and sparse standard file formats , e.g. fastahtml or similar to browser and rich standard file formats , e.g. xml , sbml ( systems biology markup language)technical documentationwritten text onlywritten text and formal structured description , e.g. automatically generated api docs ( javadoc ) , ddl ( data description language ) , dtd ( document type definition ) , uml ( unified modelling language ) , etc.written text and formal structured description and tutorials or demonstrations on how to use themdata representation standardsdata coded by local formalism onlysome data coded by a recognised controlled vocabulary , ontology or use of minimal information standards ( mibbi)general use of both recognised vocabularies or ontologies , and minimal information standards ( mibbi)data structure standardsdata structured with local model onlydata structured with formal model , e.g. an xml schemause of recognised standard model , e.g. fugeuser supportuser documentation onlyuser documentation and email / web form help desk functionuser documentation as well as a personal contact help desk function / trainingversioningno provisionprevious version of database available but no tracking of entities between versionsprevious version of database available and tracking of entities between versions the casimir database description framework ( ddf ) cabig , the nci cancer bioinformatics grid has produced a similar framework for capturing resource metadata but with a stronger focus on the technical assessment of the resources that wish to participate in the project . as cabig has a well - defined set of tasks and a user community tied to the specific vision and funding , their categories and levels are less generic than those in the ddf and more focused on assessing whether databases reach a required level of interoperability to interact with the other components of this particular project . as well as capturing the scope and database practices of resources , registries need to be explicit about the modes of programmatic access that databases provide ( e.g. web services ) as these are increasingly used to build database networks and cyberinfrastructure . this technical information is often hard to find in publications or even on database web sites , but can radically change the strategy adopted by bioinformaticians needing to access the database for example , integration into automated or semi - automated work flows using taverna such as that developed by casimir . unfortunately , traditional web - service description languages such as wsdl do not provide the required detail on the biological context of the inputs and outputs of each service to allow automated data and service integration . biocatalogue and its predecessor , the embrace service registry , address this lack of semantics by providing sites for the registration , curation , discovery and monitoring of web services for the whole biological community . curation of information about web services is open to anyone and uses a combination of free text , tags , ontology terms and example values to describe what each service does , the type of web service ( rest , soap , soaplab ) and in particular the input and outputs in terms of what type of biological data and data formats are expected . biocatalogue clearly addresses a vital requirement of the community and already some 1173 services have been annotated , despite the project only running for just over a year . having a single , well - designed solution rather than multiple competing efforts is likely to improve further uptake , and we propose that all registries of databases utilize biocatalogue to annotate the services provided by their resources rather than separately performing this task . our initial ddf metadata for over 220 resources was captured as part of a detailed mrb questionnaire sent to each resource , and active manual curation had to be used to fill in the gaps in responses . this is expensive and time - consuming and , after the first pass , there is a requirement to keep the captured data up to date , and this is not easily met . to eliminate the cost of a central curation effort , it would be much better if each resource curated their own metadata and made it accessible to the wider scientific community . as an example of this , we produced a ddf extension to the drupal content management system ( http://drupal.org ) , which allows curators to log - in and categorize their databases in terms of ddf categories and levels using a simple web form . the resulting metadata is then browsable and searchable either through a web interface or programmatically through restful web services . an example deployment is viewable at www.casimir.org.uk/casimir_ddf ( figure 1 ) and is currently populated with the metadata for the mrb project . we encourage interested readers to visit our site and for maintainers of resources to curate their metadata using it . the drupal framework is easily extensible to allow curation of other data associated with each resource , so allowing the production of a customisable community registry . the system is expected to be of great value to communities developing registry resources or individual informaticians wanting to establish quickly which features a database provides ( the software is freely available under an open - source license ) . the rest web services allow a central ddf portal to be established offering the collection and sharing of data from individual database registries as well as avoiding redundancy in curation efforts . this tool allows any user to browse a set of resources that have been annotated using the ddf categories . in addition , resource maintainers can log - in and edit their existing annotations or annotate a new resource using a simple web form . this tool is freely available and easy to install for other communities that wish to create their own registry of resources . this tool allows any user to browse a set of resources that have been annotated using the ddf categories . searches for resources by ddf category and level are also possible . in addition , resource maintainers can log - in and edit their existing annotations or annotate a new resource using a simple web form . this tool is freely available and easy to install for other communities that wish to create their own registry of resources . biocatalogue have used a combination of central and community curation from the outset to capture data on web services and the large number of services already described is testament to such an approach . again , the provision of easy to use web tools that suggest particular tags and ontology terms to use in the annotation increases the likelihood of achieving a high level of community engagement and annotation quality . communities need to acknowledge ; ( i ) a central site where they can find relevant resources would be useful , and ; ( ii ) the only practical means of achieving this is for each database to self - curate its entry using a clearly articulated and standardized set of benchmarks and tools such as provided by the ddf and biocatalogue solutions . individual resources would also benefit from this small amount of curation effort as the central registry will direct users to them , who might not previously have known about their resource . although the creators and maintainers of a resource are best placed to describe the associated metadata , a self - curation approach can raise data quality issues , but these should be minimized if the annotation tools are well designed i.e. fast and easy to use , with clear descriptions of what is being asked for , and responses presented as a lists of terms rather than free text . however , even with a well - designed annotation tool , registries are still likely to require some central curation for validating submitted data ( e.g. the ddf tool allows administrator level access to check new submissions ) . in summary , there is now a clear need for registries to be built that address biological categorization of databases and services , annotate any services provided and capture metadata on database best practises . considerable progress has been made on standardizing the capture of each of these by such approaches as the ddf and biocatalogue , but the community would benefit from coordination to produce full registries combining all these approaches . however , the value of a standard is dependent on its uptake by the community as can be seen , for example , in the mibbi family of minimal information standards . uptake of a standard is , of course , as much a social issue as one of producing the right technologies for the community . here , support from funding agencies and journals will be vital in establishing the practice of publishing database and services metadata . all curators can enhance the value of their databases by posting a minimal amount of information about their resource on a community site . the task has minimal cost , but will provide considerable value to investigators , database developers , informaticians and funding agencies . the work described above was supported by seventh framework programme of the european commission contracts to casimir ( lshg - ct-2006 - 037811 ) and enfin ( lshg - ct-2005 - 518254 ) .

the recent explosion of biological data and the concomitant proliferation of distributed databases make it challenging for biologists and bioinformaticians to discover the best data resources for their needs , and the most efficient way to access and use them . despite a rapid acceleration in uptake of syntactic and semantic standards for interoperability , it is still difficult for users to find which databases support the standards and interfaces that they need . to solve these problems , several groups are developing registries of databases that capture key metadata describing the biological scope , utility , accessibility , ease - of - use and existence of web services allowing interoperability between resources . here , we describe some of these initiatives including a novel formalism , the database description framework , for describing database operations and functionality and encouraging good database practise . we expect such approaches will result in improved discovery , uptake and utilization of data resources.database url : http://www.casimir.org.uk/casimir_ddf

Registries of databases Registries of web services Dissemination issues and solutions Funding

PMC2712784

unrelated probands of 60 families ( 30 from slovakia and 30 from the u.k . ) with a clinical phenotype suggesting a defect in gck but without a mutation in the gck coding region were included in the study . partial or entire deletions of the gck gene were previously excluded by multiplex ligation - dependent probe amplification ( mlpa ) analysis in all u.k . probands ( 9 ) . selection criteria included fpg levels 5.5 mmol / l , treatment by diet , oral hypoglycemic agents , or very low doses of insulin ( mean dose consistently < 0.3 iu kg day ) and detectable c - peptide levels . eighty - five blood relatives of the six probands with the gck pancreatic promoter mutation were subsequently contacted and invited for blood sampling and mutation testing . this study was performed with full approval of the ethics committees in bratislava and lubochna ( slovakia ) and in the u.k . 324bp of the human pancreatic islet gck promoter was amplified by pcr ( primers available upon request ) . the promoter region was examined by direct sequencing on an abi 3130 capillary sequencer ( applied biosystems , warrington , u.k . ) . sequences were compared with the published sequence ( nm_000162.2 ) , using either seqscape ( version 2.1.1 ; applied biosystems ) or mutation surveyor software ( version 3.0 ; softgenetics , cambridge , u.k . ) . haplotypes were constructed using microsatellites d7s3043 , d7s691 , d7s2428 , gck1 , gck2 , d7s667 , and d7s2506 that were run on an abi 3130 analyzer . mlpa analysis was performed in the five slovakian probands with the gck promoter mutation using the salsa mlpa kit p241-bi mody / mrc - holland ( amsterdam , the netherlands ) . results were analyzed using genemarker ( version 1.75 ; softgenetics ) . to circumvent computational constraints as a result of the pedigree size , the pedigree sk r78 was divided into 40 two - generation small pods using a facility in mega2 ( http://portal.litbio.org/registered/help/mega2/index/html ) and the analyses were run in merlin ( 12 ) . the logarithm of odds ( lod ) score for family sk r78 was performed applying a rare autosomal dominant inheritance model using a disease allele frequency of 0.001 and a frequency of phenocopies of 0.1% . fasting venous blood samples for glucose and hormonal analyses were collected into edta tubes ( sarstedt , nmbrecht , germany ) . plasma glucose concentrations were measured with the glucose oxidase method ( hitachi 911 , hitachinaka , japan ) . insulin and c - peptide were determined using the elecsys ( roche , switzerland ) chemiluminescence automatic analyzer . alignment of the human -cell gck promoter was performed using the university of california santa cruz ( uscs ) bioinformatics blat tool . the human -cell promoter was analyzed for potential transcription factor binding sites by running a matrix search for transcription factor binding sites ( match ) using transfac professional ( version 12.1 ) . pcr primers were designed to generate five different lengths of the human gck upstream promoter sequence ( supplementary tables and figures , available in an online appendix at http//diabetes.diabetesjournal.org / cgi / content / full / db09 - 0070/dc1 ) . the promoter fragments were amplified from human genomic dna and subcloned into the pcr 2.1 topo vector ( invitrogen , paisley , u.k . ) . prior to cloning , dna sequencing was performed to ensure that the -cell gck promoter haplotype associated with variation in fpg levels was not included ( 13 ) . the fragments were subcloned into a predigested pgl3-basic luciferase reporter vector ( promega , southampton , u.k . ) . the 71g > c mutation was introduced using a quick change mutagenesis kit ( stratagene , la jolla , ca ) ( supplementary table 2 ) . ins-1 cells were cultured as previously described to a density of 1 10 per well in 24-well tissue culture dishes 24 h before transfection ( 14 ) . cotransfection was performed using lipofectamine reagent ( invitrogen ) with 580 ng pgl3 promoter construct dna plus 20 ng prl - tk per well , according to the manufacturer 's protocol . transfected cells were harvested after 24 h and assayed for luciferase activity in a veritas microplate luminometer using the dual luciferase reporter assay system ( promega ) . the renilla luciferase ( prl - tk ) was used as a recovery marker for data normalization . promoter data are reported as the ratio of the firefly / renilla arbitrary units for each sample minus the value gained by the promoterless pgl3-basic vector . both wild - type and 71 mutant gck promoter oligonucleotides were generated spanning the potential sp1 binding region predicted from transfac analysis and corresponding to sequences 53 to 88bp from the transcriptional start site . an additional oligonucleotide corresponding to a known sp1 binding site was also used as a positive control for sp1 binding ( promega ) . binding reactions and electrophoretic mobility shift assays were conducted as previously described ( 15,16 ) . briefly , 200 ng sp1 protein ( promega ) was incubated with 0.20.5 ng of 32p - labeled probe ( 15 10 cpm ) at room temperature for 30 min . for supershift or competition analysis , the reaction mixture was preincubated with 1 g sp1 or usf1 antiserum ( santa cruz biotechnology , heidelberg , germany ) or with 7.5 pmol ( 100x ) unlabeled oligonucleotide probe at room temperature for 20 min prior to addition of the labeled probe . 324bp of the human pancreatic islet gck promoter was amplified by pcr ( primers available upon request ) . the promoter region was examined by direct sequencing on an abi 3130 capillary sequencer ( applied biosystems , warrington , u.k . ) . sequences were compared with the published sequence ( nm_000162.2 ) , using either seqscape ( version 2.1.1 ; applied biosystems ) or mutation surveyor software ( version 3.0 ; softgenetics , cambridge , u.k . ) . haplotypes were constructed using microsatellites d7s3043 , d7s691 , d7s2428 , gck1 , gck2 , d7s667 , and d7s2506 that were run on an abi 3130 analyzer . mlpa analysis was performed in the five slovakian probands with the gck promoter mutation using the salsa mlpa kit p241-bi mody / mrc - holland ( amsterdam , the netherlands ) . results were analyzed using genemarker ( version 1.75 ; softgenetics ) . to circumvent computational constraints as a result of the pedigree size , the pedigree sk r78 was divided into 40 two - generation small pods using a facility in mega2 ( http://portal.litbio.org/registered/help/mega2/index/html ) and the analyses were run in merlin ( 12 ) . the logarithm of odds ( lod ) score for family sk r78 was performed applying a rare autosomal dominant inheritance model using a disease allele frequency of 0.001 and a frequency of phenocopies of 0.1% . fasting venous blood samples for glucose and hormonal analyses were collected into edta tubes ( sarstedt , nmbrecht , germany ) . plasma glucose concentrations were measured with the glucose oxidase method ( hitachi 911 , hitachinaka , japan ) . insulin and c - peptide were determined using the elecsys ( roche , switzerland ) chemiluminescence automatic analyzer . alignment of the human -cell gck promoter was performed using the university of california santa cruz ( uscs ) bioinformatics blat tool . the human -cell promoter was analyzed for potential transcription factor binding sites by running a matrix search for transcription factor binding sites ( match ) using transfac professional ( version 12.1 ) . pcr primers were designed to generate five different lengths of the human gck upstream promoter sequence ( supplementary tables and figures , available in an online appendix at http//diabetes.diabetesjournal.org / cgi / content / full / db09 - 0070/dc1 ) . the promoter fragments were amplified from human genomic dna and subcloned into the pcr 2.1 topo vector ( invitrogen , paisley , u.k . ) . prior to cloning , dna sequencing was performed to ensure that the -cell gck promoter haplotype associated with variation in fpg levels was not included ( 13 ) . the fragments were subcloned into a predigested pgl3-basic luciferase reporter vector ( promega , southampton , u.k . ) . the 71g > c mutation was introduced using a quick change mutagenesis kit ( stratagene , la jolla , ca ) ( supplementary table 2 ) . ins-1 cells were cultured as previously described to a density of 1 10 per well in 24-well tissue culture dishes 24 h before transfection ( 14 ) . cotransfection was performed using lipofectamine reagent ( invitrogen ) with 580 ng pgl3 promoter construct dna plus 20 ng prl - tk per well , according to the manufacturer 's protocol . transfected cells were harvested after 24 h and assayed for luciferase activity in a veritas microplate luminometer using the dual luciferase reporter assay system ( promega ) . the renilla luciferase ( prl - tk ) was used as a recovery marker for data normalization . promoter data are reported as the ratio of the firefly / renilla arbitrary units for each sample minus the value gained by the promoterless pgl3-basic vector . both wild - type and 71 mutant gck promoter oligonucleotides were generated spanning the potential sp1 binding region predicted from transfac analysis and corresponding to sequences 53 to 88bp from the transcriptional start site . an additional oligonucleotide corresponding to a known sp1 binding site was also used as a positive control for sp1 binding ( promega ) . binding reactions and electrophoretic mobility shift assays were conducted as previously described ( 15,16 ) . briefly , 200 ng sp1 protein ( promega ) was incubated with 0.20.5 ng of 32p - labeled probe ( 15 10 cpm ) at room temperature for 30 min . for supershift or competition analysis , the reaction mixture was preincubated with 1 g sp1 or usf1 antiserum ( santa cruz biotechnology , heidelberg , germany ) or with 7.5 pmol ( 100x ) unlabeled oligonucleotide probe at room temperature for 20 min prior to addition of the labeled probe . screening of 60 probands with a gck - mody phenotype and no abnormality of the gck coding sequence identified six ( five from slovakia and one from the u.k . ) apparently unrelated individuals with a novel 71g > c mutation . mmol / l ) in a total of 39 affected individuals and 52 unaffected individuals across the six pedigrees ( fig . the lod score in family sk r78 alone was 5.00 that is well above the proposed guidelines for claiming statistically significant linkage with a lod score of > 3.0 ( 17 ) . haplotype analysis in the u.k . family and in four of the slovakian families demonstrated that the mutation had arisen independently and there was no evidence for a founder effect ( fig . the -cell 71g > c promoter mutation is responsible for 30% ( 5 of 17 families ) of known cases of gck - mody in slovakia ( d.g . , j.s . , and i.k . , unpublished data ) , whereas in the u.k . only one family has been identified and over 160 gck - mody cases with coding mutations have been identified ( k.c . , a : extended pedigrees of the six probands with the novel 71g > c gck promoter mutation showing five slovakian ( sk r78 , sk r25 , sk r71 , sk r94 , and sk r124 ) and one u.k . individuals v:9 ( sk r78 family ) and i:2 ( sk r25 family ) are diabetic subjects ( phenocopies ) with the nn genotype ( see results ) . nm , heterozygous for the 71g > c gck promoter mutation ; nn , wild type . b : haplotype analysis performed in five of the six families with the novel gck promoter mutation . squares denote male subjects , circles denote female subjects , and solid symbols show individuals with fasting hyperglycemia ( 5.5 solid bars indicate the haplotype cosegregating with fasting hyperglycemia on which the gck c.71g > c mutation has arisen . the marker order , location of the gck gene , and location on chromosome 7 are shown in the box . the clinical characteristics of the individuals with the novel promoter mutation are summarized in table 1 . in line with other cases of gck - mody , all patients had fasting hyperglycemia ( 8) . the age at diagnosis ranged from 6 to 81 years ; 15 case subjects were identified following the detection of the gck mutation in one of the six probands . in affected individuals , bmi ranged from 18.2 to 40.8 kg / m with a mean sd value of 24.6 5.3 kg / m . the majority of patients ( 23 of 39 [ 59% ] ) with the mutation are registered with and seen by a diabetologist . clinical characteristics of individuals with the novel 71g > c mutation data are means ( range ) or means sd unless otherwise indicated . of these , 83% ( 19 of 23 ) are managed by diet alone , with three patients on oral hypoglycemic agents ( one receiving metformin [ 1,000 mg / day ] and two receiving sulfonylureas [ diaprel mr , twice daily ] ) and one patient on insulin treatment ( nph insulin [ 0.22 units kg day ] ) . none of these patients have had a1c values > 7% , whereas a1c values on treatment are all < 6.5% . fpg levels in these patients are independent of insulin dose and/or oral hypoglycemic agents used . one patient , a female aged 50 years ( pedigree sk r78 , subject v:9 ) ( fig . 1a ) with a bmi 28.0 kg / m has several features consistent with the metabolic syndrome including hypertension ( blood pressure 140/90 mmhg ) and dyslipidemia ( total cholesterol 5.79 mmol / l ) . she was diagnosed with hyperglycemia at the age of 47 years and is currently treated with metformin . the second female patient ( sk r25 , i:2 ) ( fig . 1a ) was diagnosed with diabetes at the age of 49 years and is insulin treated . no further clinical or laboratory data are available on this subject because following blood withdrawal for genetic testing the patient refused to cooperate further in the study . based on these clinical characteristics , both of these patients have a phenotype that is consistent with classical type 2 diabetes and can be considered as phenocopies . to establish the appropriate promoter construct in which to study the effect of the 71g > c mutation , reporter gene constructs containing different gck promoter fragment lengths were prepared ranging from 263bp to 1031bp with respect to the atg start site . . a : comparison of luciferase activity generated with varying lengths of upstream gck promoter . the promoter fragment lengths incorporated into the pgl3-basic vector are shown on the left of the figure and ranged from 263bp to 1031bp . the bar chart on the right shows mean se luciferase activity in cotransfected ins-1 cells with the different promoter lengths . b : functional characterization of the novel gck 71g > c promoter mutation demonstrates reduced promoter activity . mean luciferase activity of wild - type promoter fragments of 430bp and 1031bp is represented by the black bars , whereas the luciferase activity of their counterparts containing a 71g > c mutation is represented by the white bars se . the reduction in activity was significant ( * p = 3.6 10 and * * p = 1.9 10 ) . expression is in ins-1 cells , and reported luciferase activity is normalized to renilla and pgl3 null expression levels . decreased expression levels were seen with fragments 618bp and 826bp suggesting the presence of repressor elements within this region . the effect of the 71g > c mutation was then analyzed using the 430bp promoter fragment that had near - maximal reporter gene expression , indicating that necessary binding sites for expression were contained in this fragment ; site - directed mutagenesis was also carried out for the full length 1031bp fragment . when these constructs were analyzed , the gck 71g > c promoter variant caused a dramatic reduction in promoter activity in the presence of the c versus g allele for both promoter lengths ( p = 3.6 10 and 1.9 10 for the 430bp and 1031bp fragments , respectively ) ( fig . this provides robust evidence that the point mutation has a repressive effect on gene expression , suggesting either allele - specific recruitment of a transcriptional repressor or loss of activator binding . using the uscs blat tool , no sequences could be identified corresponding to the 64bp to 89bp region of the human gck promoter in the mouse or rat gck promoters as a result of either a deletion in the rodent or an insertion in the human dna ( supplementary fig . match analysis conducted on the wild - type gck promoter identified a number of potential activator and repressor regions within the 71bp region of interest ( supplementary table 4 ) . identical match analysis conducted on the 71 variant gck promoter demonstrated the potential loss of the majority of these transcription factor binding sites including the transcriptional activator sp1 ( supplementary table 4 ) . to demonstrate that sp1 can indeed bind to the putative binding site located at the 71bp region of the wild - type promoter , gel shift experiments were performed . using the wild - type oligonucleotide , two clear bands that were specific on competition with molar excess of unlabeled self or an unlabeled probe corresponding to a consensus sp1 binding site sp1 binds both the wild - type and sp1 oligonucleotides , whereas the 71g > c mutation causes a dramatic reduction in sp1 binding . a : labeled gck promoter ( 53 to 88 ) and sp1 consensus oligonucleotides were incubated in the presence or absence of sp1 nuclear protein . b : specific sp1 and nonspecific usf antibodies were used to confirm the specificity of sp1 binding that was visualized as a supershifted band of reduced mobility . the mobility of these bands matched those seen when the consensus sp1 binding site probe was radiolabeled and used in the binding reaction ( fig . the complexes were lost when a radiolabeled probe corresponding to the c - allele of the point mutation was used in the binding reaction ; this probe also competed much less effectively when used as an unlabeled competitor ( fig . recruitment of sp1 was confirmed by supershift experiments for both the sp1 consensus probe and the 71 wild - type probe bearing the g - allele ( fig . screening of 60 probands with a gck - mody phenotype and no abnormality of the gck coding sequence identified six ( five from slovakia and one from the u.k . ) apparently unrelated individuals with a novel 71g > c mutation . mmol / l ) in a total of 39 affected individuals and 52 unaffected individuals across the six pedigrees ( fig . the lod score in family sk r78 alone was 5.00 that is well above the proposed guidelines for claiming statistically significant linkage with a lod score of > 3.0 ( 17 ) . haplotype analysis in the u.k . family and in four of the slovakian families demonstrated that the mutation had arisen independently and there was no evidence for a founder effect ( fig . the -cell 71g > c promoter mutation is responsible for 30% ( 5 of 17 families ) of known cases of gck - mody in slovakia ( d.g . , j.s . , and i.k . , unpublished data ) , whereas in the u.k . only one family has been identified and over 160 gck - mody cases with coding mutations have been identified ( k.c . , a : extended pedigrees of the six probands with the novel 71g > c gck promoter mutation showing five slovakian ( sk r78 , sk r25 , sk r71 , sk r94 , and sk r124 ) and one u.k . individuals v:9 ( sk r78 family ) and i:2 ( sk r25 family ) are diabetic subjects ( phenocopies ) with the nn genotype ( see results ) . nm , heterozygous for the 71g > c gck promoter mutation ; nn , wild type . b : haplotype analysis performed in five of the six families with the novel gck promoter mutation . squares denote male subjects , circles denote female subjects , and solid symbols show individuals with fasting hyperglycemia ( 5.5 solid bars indicate the haplotype cosegregating with fasting hyperglycemia on which the gck c.71g > c mutation has arisen . the marker order , location of the gck gene , and location on chromosome 7 are shown in the box . the clinical characteristics of the individuals with the novel promoter mutation are summarized in table 1 . in line with other cases of gck - mody , all patients had fasting hyperglycemia ( 8) . the age at diagnosis ranged from 6 to 81 years ; 15 case subjects were identified following the detection of the gck mutation in one of the six probands . in affected individuals , bmi ranged from 18.2 to 40.8 kg / m with a mean sd value of 24.6 5.3 kg / m . the majority of patients ( 23 of 39 [ 59% ] ) with the mutation are registered with and seen by a diabetologist . clinical characteristics of individuals with the novel 71g > c mutation data are means ( range ) or means sd unless otherwise indicated . of these , 83% ( 19 of 23 ) are managed by diet alone , with three patients on oral hypoglycemic agents ( one receiving metformin [ 1,000 mg / day ] and two receiving sulfonylureas [ diaprel mr , twice daily ] ) and one patient on insulin treatment ( nph insulin [ 0.22 units kg day ] ) . none of these patients have had a1c values > 7% , whereas a1c values on treatment are all < 6.5% . fpg levels in these patients are independent of insulin dose and/or oral hypoglycemic agents used . one patient , a female aged 50 years ( pedigree sk r78 , subject v:9 ) ( fig . 1a ) with a bmi 28.0 kg / m has several features consistent with the metabolic syndrome including hypertension ( blood pressure 140/90 mmhg ) and dyslipidemia ( total cholesterol 5.79 mmol / l ) . she was diagnosed with hyperglycemia at the age of 47 years and is currently treated with metformin . the second female patient ( sk r25 , i:2 ) ( fig . 1a ) was diagnosed with diabetes at the age of 49 years and is insulin treated . no further clinical or laboratory data are available on this subject because following blood withdrawal for genetic testing the patient refused to cooperate further in the study . based on these clinical characteristics , both of these patients have a phenotype that is consistent with classical type 2 diabetes and can be considered as phenocopies . to establish the appropriate promoter construct in which to study the effect of the 71g > c mutation , reporter gene constructs containing different gck promoter fragment lengths were prepared ranging from 263bp to 1031bp with respect to the atg start site . . a : comparison of luciferase activity generated with varying lengths of upstream gck promoter . the promoter fragment lengths incorporated into the pgl3-basic vector are shown on the left of the figure and ranged from 263bp to 1031bp . the bar chart on the right shows mean se luciferase activity in cotransfected ins-1 cells with the different promoter lengths . b : functional characterization of the novel gck 71g > c promoter mutation demonstrates reduced promoter activity . mean luciferase activity of wild - type promoter fragments of 430bp and 1031bp is represented by the black bars , whereas the luciferase activity of their counterparts containing a 71g > c mutation is represented by the white bars se . the reduction in activity was significant ( * p = 3.6 10 and * * p = 1.9 10 ) . expression is in ins-1 cells , and reported luciferase activity is normalized to renilla and pgl3 null expression levels . decreased expression levels were seen with fragments 618bp and 826bp suggesting the presence of repressor elements within this region . the effect of the 71g > c mutation was then analyzed using the 430bp promoter fragment that had near - maximal reporter gene expression , indicating that necessary binding sites for expression were contained in this fragment ; site - directed mutagenesis was also carried out for the full length 1031bp fragment . when these constructs were analyzed , the gck 71g > c promoter variant caused a dramatic reduction in promoter activity in the presence of the c versus g allele for both promoter lengths ( p = 3.6 10 and 1.9 10 for the 430bp and 1031bp fragments , respectively ) ( fig . this provides robust evidence that the point mutation has a repressive effect on gene expression , suggesting either allele - specific recruitment of a transcriptional repressor or loss of activator binding . using the uscs blat tool , no sequences could be identified corresponding to the 64bp to 89bp region of the human gck promoter in the mouse or rat gck promoters as a result of either a deletion in the rodent or an insertion in the human dna ( supplementary fig . 1 ) . match analysis conducted on the wild - type gck promoter identified a number of potential activator and repressor regions within the 71bp region of interest ( supplementary table 4 ) . identical match analysis conducted on the 71 variant gck promoter demonstrated the potential loss of the majority of these transcription factor binding sites including the transcriptional activator sp1 ( supplementary table 4 ) . to demonstrate that sp1 can indeed bind to the putative binding site located at the 71bp region of the wild - type promoter , gel shift experiments were performed . using the wild - type oligonucleotide , two clear bands that were specific on competition with molar excess of unlabeled self or an unlabeled probe corresponding to a consensus sp1 binding site sp1 binds both the wild - type and sp1 oligonucleotides , whereas the 71g > c mutation causes a dramatic reduction in sp1 binding . a : labeled gck promoter ( 53 to 88 ) and sp1 consensus oligonucleotides were incubated in the presence or absence of sp1 nuclear protein . b : specific sp1 and nonspecific usf antibodies were used to confirm the specificity of sp1 binding that was visualized as a supershifted band of reduced mobility . the mobility of these bands matched those seen when the consensus sp1 binding site probe was radiolabeled and used in the binding reaction ( fig . the complexes were lost when a radiolabeled probe corresponding to the c - allele of the point mutation was used in the binding reaction ; this probe also competed much less effectively when used as an unlabeled competitor ( fig . recruitment of sp1 was confirmed by supershift experiments for both the sp1 consensus probe and the 71 wild - type probe bearing the g - allele ( fig . in this study , we have identified the first mutation in the gck -cell promoter that causes elevated glucose levels as a result of decreased gck expression in six probands : five from slovakia and one from the u.k . the mutation cosegregated with fasting hyperglycemia in all six families with a total of 39 affected individuals ( fig . and slovakian families , with no evidence for a founder effect in the slovakian families ( fig . the clinical phenotype of patients with gck - mody attributable to a promoter mutation is indistinguishable from that caused by a mutation in the coding region of the gene ( table 1 and ref . this is entirely expected as compensation will be provided by the wild - type allele ( 18,19 ) . functional characterization of the gck promoter clearly demonstrates that the gck 71g > c promoter variant causes a dramatic reduction in promoter activity . bioinformatic analysis predicted that this mutation could potentially result in the loss of transcription factor binding within the 71bp region . allele - specific modulation of sp1 binding was then demonstrated by the electrophoretic mobility shift assay . sp1 has previously been demonstrated to enhance gck transcription in gilthead sea bream ( sparus aurata ) ( 20 ) . in rodent studies , sp1 has also been shown to play an integral part in the mechanisms for the transcription of several glycolytic enzymes , such as pyruvate kinase , aldolase a , and acetyl - coa ( 2123 ) . the results clearly show an allele - specific difference in sp1 binding with the 71g > c mutation losing sp1 binding capacity . this provides a clear explanation of the reduced gene expression levels associated with this novel mutation and the first evidence in humans for an essential role of sp1 regulation in gck expression . the significance of this finding is that routine molecular diagnostic testing for gck - mody does not currently include the promoter region . given the implications for prognosis , treatment decisions , and inheritance , it is important to identify these individuals ( 24 ) . interestingly , a comparison of the promoter sequences across species demonstrated that the sequence containing the novel mutation was absent from both the rat and mouse -cell promoters . this is unusual because normally pathogenic mutations occur in regions that are conserved ( and therefore supposed to be functionally important ) across species . this observation has implications for the design of future studies evaluating the role of noncoding regions in the pathogenesis of both monogenic and multifactorial diabetes . we have identified the first mutation in the gck -cell promoter in a total of 39 individuals with mild fasting hyperglycemia . this mutation has been demonstrated at the genetic and functional level to cause gck - mody . our results support the inclusion of the gck -cell promoter region in routine diagnostic testing for gck - mody .

objectiveinactivating mutations in glucokinase ( gck ) cause mild fasting hyperglycemia . identification of a gck mutation has implications for treatment and prognosis ; therefore , it is important to identify these individuals . a significant number of patients have a phenotype suggesting a defect in glucokinase but no abnormality of gck . we hypothesized that the gck -cell promoter region , which currently is not routinely screened , could contain pathogenic mutations ; therefore , we sequenced this region in 60 such probands.research design and methodsthe -cell gck promoter was sequenced in patient dna . the effect of the identified novel mutation on gck promoter activity was assessed using a luciferase reporter gene expression system . electrophoretic mobility shift assays ( emsas ) were used to determine the impact of the mutation on sp1 binding.resultsa novel 71g > c mutation was identified in a nonconserved region of the human promoter sequence in six apparently unrelated probands . family testing established cosegregation with fasting hyperglycemia ( 5.5 mmol / l ) in 39 affected individuals . haplotype analysis in the u.k . family and four of the slovakian families demonstrated that the mutation had arisen independently . the mutation maps to a potential transcriptional activator binding site for sp1 . reporter assays demonstrated that the mutation reduces promoter activity by up to fourfold . emsas demonstrated a dramatic reduction in sp1 binding to the promoter sequence corresponding to the mutant allele.conclusionsa novel -cell gck promoter mutation was identified that significantly reduces gene expression in vitro through loss of regulation by sp1 . to ensure correct diagnosis of potential gck - mody ( maturity - onset diabetes of the young ) cases , analysis of the -cell gck promoter should be included .

RESEARCH DESIGN AND METHODS Genetic analysis of the human -cell Biochemistry. -Cell Transfection constructs. Luciferase assay. Electrophoretic mobility shift assays. RESULTS Identification of a novel 71G>C Clinical characteristics of patients with the novel promoter mutation. Activity of -cell Identification of potential transcription factor binding sites in the -cell Sp1 binding is reduced due to the 71G>C mutation. DISCUSSION Supplementary Material

PMC4810916

adhesion is defined as the process of cross - linking between an adhesive and a solid or liquid substrate . close contact between the adhesive and the substrate is essential in achieving the strong adhesion . in addition , the surface tension of adhesive should be less than the free energy of substrate surface . adhesion in the etch - and - rinse systems is based on infiltration of the resin between the unsupported collagen fibers , by removing the smear layer and then production of a hybrid layer . the spatial structure and arrangement of the collagen fibers influence the interfiber spaces and the efficiency of primer penetration into those spaces . the self - etching systems are based on etching and contemporary penetration of acidic monomers . acidic monomers do not dissolve the smear layer and the mineral components of dentin completely . substrate characteristics and thickness , tensions in the resin - dentin interface , and hybrid layer characteristics affect bonding to dentin . higher bond strengths between resin and dentin can be achieved using different types of dentin bonding agents , using appropriate clinical methods such as multiple adhesive coatings , increasing the application time of bonding agents , delaying polymerization , and longer exposure times of the bonding system . most of these methods result in a higher quality hybrid layer and a stronger polymer . temperature has been reported as an effective factor on bond strength of composites to the dental tissue . many studies have shown that different temperatures of the dentin bonding agent , drying air spray , restorative material and environment have on bond strength of resin composite to dentin . they showed that the increase or decrease in temperature affects the bond strength of composite to dentin . heat increases the kinetic energy of molecules which can affect characteristics of the bonding agent and the substrate . it has been demonstrated that warm air drying evaporates the bonding agent solvent , resulting in stronger dentin - resin bonding . in addition , warming the bonding agent has been reported to increase the bonding strength . since the washing is an essential part of the adhesion process , it seems that the washing water temperature can be used to affect the temperature of the substrate ( i.e. , enamel or dentin ) . the higher temperature may be able to decrease the bonding agent viscosity , which could lead to better penetration of bonding agent into both etched enamel and the exposed collagen in dentin and improve tooth - composite adhesion . the purpose of this study was to evaluate the effect of different temperatures of irrigation water on micro - shear bond strength ( sbs ) of a two - step self - etching primer and a two - step etch - and - rinse adhesive system . the null hypothesis was that sbs was not affected by the temperature of washing water in both of the bonding systems . in this in vitro study , 24 intact human third molars were used which were extracted within a period of 3 months before the experiment . the teeth were washed and cleaned under running water immediately and stored in a 0.2% thymol solution at room temperature ( 23c ) . the teeth were individually embedded in self - cure orthodontic resin ( orthoresin , dentsply ltd . , weybridge , england ) with the buccal and lingual surfaces of crowns oriented toward the external block face to expose the buccal and lingual enamel . flat dentin surfaces were created on mid - buccal and mid - lingual of each sample , using a low - speed diamond saw ( 180 grit dandiran , iran ) , under running water coolant . the exposed dentin surfaces were polished with # 600-grit silicone - carbide paper for 60 s under running water to create a standard smear layer . the samples were divided into three experimental groups ( 5 2c , 22 2c and 50 2c ) with eight teeth in each group . then , they were stored in distilled water at room temperature . to set the washing water temperature to 5 2c , eight 50 ml syringes filled with distilled water were refrigerated at 5c for 24 h before the experiment . to make hot water for the experiment , a calibrated compound heater ( hanau uh-1 115v vintage dental lab utility heater , usa ) was used . the adhesive bonding agents , which were a two - step self - etching primer ( se , clearfil se bond kuraray medical inc . , okoyama , japan ) and a two - step etch - and - rinse adhesive ( single bond [ sb ] , 3 m espe , st . paul , mn , usa ) [ table 1 ] , were stored in the temperature the manufactures recommended ( 5 2c ) until an hour before the experiments . in the se group , dentin surfaces were rinsed with distilled water at warm ( 50 2c ) , cold ( 5 2c ) or room temperature ( 22 2c ) for 20 s , and then blotted dry with a cotton pellet to reduce the temperature fluctuations . the surface temperature was measured with a digital thermometer with 94% accuracy ( scantemp 485 , dostmann electronic , china ) [ table 2 ] . adhesive systems : compositions and batch number adhesive systems : application mode , water , and dentin surface temperature in se groups , the self - etching primer was applied to the surface by micro brush for 20 s. the primed surface was gently air - dried with 0.15 mpa oil - free compressed air and temperature of 21c . the air syringe was kept at a distance of 10 cm from the sample for 5 s. then , the bonding agent was applied on the surface , following the manufacturer 's instructions , and then gently air - dried [ table 2 ] . bonding procedure for sb groups was done on the other side of all teeth at the same temperature . after acid etching with 35% phosphoric acid gel ( ultra - etch , ultradent products inc . , south jordan , ut , usa ) , the respective etchant for 20 s , the dentin surfaces were rinsed for 10 s , and then compressed air and water for another 10 s. in this stage , the samples were rinsed with distilled water with various temperatures for 20 s and dried with cotton pellets . two coats of adhesive were used on the surfaces ; the first layer was agitated for 15 s and then air - dried at temperature of 21c for 5 s at a distance of 10 cm . the second layer was used and dried gently . prior to light curing of bonding resin in each specimen , two hollow cylinders from micro - bore tygon tube ( norton performance plastic , oh , usa ) with 1 mm height and an internal diameter of 0.8 mm were placed on the mesial and distal ( 16 cylinders for each bonding agent at each temperature ) of treated dentin surfaces . the adhesive resins were light - cured with a halogen light cure unit ( coltlux 75 , coltene , switzerland ) , with a power density of 1000 mw / cm for 10 s. resin composite ( filtek z250 , shadea2 , 3 m espe , st . paul , mn , usa ) was carefully inserted into the cylinders and was subjected to curing light for 40 s , during which the light was moved to assure curing of the entire cylinder . after an hour of storing the specimens in distilled water at 37c , the tygon tubes were cut and removed gently . the specimens were then incubated for 48 h in distilled water and thermo cycling ( deltatpo2 , nemo , mashhad , iran ) was done ( 1000 cycles between 5c and 55c ) with a dwell time of 30 s in each bath and a transfer time of 5 - 10 s between the two baths . the specimens were fixed in a universal testing machine ( nonstop , bego , germany ) . a thin steel wire ( 0.275 mm d ) was looped flush between the load cell projection and the composite cylinder to make contact with the lower half - circle of the cylinder and stay parallel to the loading cell movement direction . the loop was in line with the force vector and perpendicular to the dentin - resin interface to apply shear force . force was applied with wire loop at a crosshead speed of 1 mm / min until failure in the composite cylinder occurred . the maximum load at the time of failure was recorded in kilograms ( kg ) , which was converted to mpa for each specimen . the mode of failure was determined at 40 using a stereomicroscope ( leicams5 , wetzlar , germany ) and recorded as adhesive failure ( a ) , cohesive ( c ) , and mixed modes ( m ) on the same fractured surface . the bond strength results were analyzed using a two - way analysis of variance ( anova ) to detect the overall effect of the test factors ( water temperature and bonding systems ) . one - way anova and tukey 's post hoc test were used to determine the effect of temperature on bond strength . paired sample t - test was used to compare the bond strength between two types of adhesive at the same temperature , and failure mode distributions were analyzed by fisher excat test . two - way anova analysis showed that sbs was significantly influenced by temperature ( p = 0.022 , f = 3.981 ) and the interactions of temperature and type of adhesive system ( p = 0.013 , f = 4.602 ) . sbs was not affected by type of adhesive system ( p = 0.261 , f = 1.283 ) . micro - shear bond strength mean values standard deviation in mpa and fracture mode distribution one - way anova and post hoc tukey 's tests showed that in the sb groups , there were significant differences between sbs of the warm washing water group ( 50c ) and sbs of 5c and 22c washing water temperature groups ( p = 0.003 and p = 0.019 , respectively ) . no statistical difference was observed between the cold water group and the room temperature water groups . there were no statistical differences between three different water temperatures of the se . according to the paired sample t - test , the sbs was higher in the sb at 50 2c compared with se ( p = 0.007 ) . also , the sbs in the se was significantly higher than the sb at 22 2c ( p = 0.031 ) . although higher bond strength in cold water was observed for the se , difference was not significant . fisher exact test showed no significant difference between different groups which means water temperature and bonding agent had no effect on fracture modes . it is shown in this study that temperature increase can impact the substrate , dentin , or smear layer , depending on the adhesive system . in addition , temperature rise can change the physical properties and the chemical structure of the materials . the etch - and - rinse systems , such as sb , bond to dentin by removing the smear layer and penetrating into the collagen mesh of the resin . mild self - etching systems such as se , on the other hand , are only capable of dissolving the smear layer and simultaneously penetrating the resin by etching the surface . the findings of this paper showed that the bond strength of se at both low temperature ( 5 2c ) and room temperature of washing water ( 22 2c ) is more than sb bond strength . se contains 10-methacryloxydecyldihydrogen phosphate ( 10-mdp ) as a functional monomer that appears not only to interact more intensively with hydroxyapatite ( hap ) but also to have the most hydrolytically stable bond with calcium . this interaction is more stable than the interaction caused by the long term connection of 10-mdp and hap . since the calcium salt of the acidic molecule is less soluble , the molecular adhesion to hap of substrate is more stable and intense . in addition , the hybrid layer has a substantially higher modulus for the self - etching systems . this may be a possible explanation for the higher bond strength of these materials . also , use of phosphoric acid in high concentrations ( 35% and upper ) causes denaturation of the top layer of collagen , which causes the lower bond strength of sb . in the present study , the bond strength of sb was increased with increases in the washing water temperature and it was higher at 50 2c compared to that of se . it should be noted that in etch - and - rinse systems , after acid etching and demineralization , the collagen fibers collapse and interpeptidal hydrogen bonds are generated that cause more collagen mesh shrinkage . the hydrogen bond is broken with hydroxyethyl methacrilate ( hema ) and water molecules during resin adhesion . moreover , a demineralized dentin zone at the bottom of the hybrid layer created by insufficient infiltration of resin is thought to be the site where interfacial degradation is expected to begin . heat can reduce the effect of these phenomena by both affecting the etched dentin surface and changing the physical properties of the bonding agent . based on previous studies , the hydrogen bond strength decreases and the hydrogen bond length increases as a result of an increase in the temperature . these mean that the unfolding of the helix and breaking of the bond by hema and water occur more easily . in addition , heat increases the free energy of surface that decreases the contact angle of surface - liquid , which in turn facilitates the liquid diffusion and infiltration . the increase in the temperature might enable free radicals and propagate polymer chains to become more flexible as a result of decreased viscosity . the higher temperature could also lessen the amount of bounded water retained in the resin . reported that an increase of 8c in the temperature increased the spreading velocity of the sb and prime and bond nt by approximately 21% due to decreased viscosity at high temperature . all of these impacts help a better penetration and diffusion of the resin that reduces the demineralized zone . this consequently leads to better micromechanical lock and higher bond strength of resin - dentin . showed the blow of the warm air causes the sb solvent evaporation , which increases the resin - dentin bond strength . since sb is a water / ethanol - based system , the duration of heat transfer for effective evaporation of the solvent is not enough compared to direct air blowing . for se groups , at first , the ascending or descending alteration of the bond strength seemed to be associated with increase in temperature , but the higher bond strength at room temperature compared to low and high temperatures was observed . it seems the bond strength variations induced by the various temperatures in the self - etching system such as se is a combination of the effects of alteration in substrate , acidic monomer and their interaction . some experiments reported that the smear layer thickness is not effective on the function of self - etching systems , but other studies suggested that the smear layer thickness interferes with the self - etching bond strength . it seems the smear layer effect is associated with its density which affects the primer 's ability to etch through the smear layer . although burs cutting forces generate friction and heat which make the gelatin status of modified collagen , the sole effect of temperature increase was not evaluated up to now . thus , the gelatin status of the smear layer may increase by an increase in temperature and leads to difficult penetration of primer into this layer . the resin infiltrates into the demineralized dentin and retains hap crystals around collagen fibers , and therefore prevents collagen fibers from being stained . moreover , the less soluble calcium salt causes the acidic molecule adhesion to hap of substrate to be more stable and intense . this reaction happens at room temperature , under high buffering ability of the smear layer neutralizes the acidic monomers partially and the mixture 's ph values increases . this increase in ph may prevent more dissolution of hap . with an increased temperature , in this situation , acidity of environment is more than the acidity at room temperature . likely , with an increase in the acidity of the environment , hap around collagen fibers dissolves and degradation of the bond may occur . moreover , the interaction of acidic monomer and calcium for producing calcium salt is an exothermal reaction . at higher temperature , the participative molecules in exothermal reaction have less likelihood to combine and this leads to less production of calcium salt . the reduced calcium salt level may result in difficulty in the stability of resin - dentin bond . although some researches have attempted to explain the effect of increase rate and pressure of evaporation , on reduction of demineralization ability of adhesive , those results were not supported by garcia et al . experiment . furthermore , the solvent in the se is water , which needs more time and warmer temperature for enough evaporation . low temperature causes more buffering ability of the smear layer and reduction in the surface free energy . low surface free energy reduces resin penetration into smear layer and hap 's insufficient wetting . two important issues that should be considered are the effect of high temperature on pulp vitality , and dentinal fluid flow . it seems the warmth of high temperature water , measured at pulp side of dentin , is not higher than the warmth caused by curing units in composite 's polymerization . quartz tungsten halogen curing unit raises the temperature in resin - based composite to approximately 15.5 - 18.6c . the higher intrapulpal temperature ( 8c ) for 10 s was reported during the application of a plasma arc lamp , with remaining dentin thickness of 1 mm . with considering to hydrodynamic theory during application of the warm water on the surface , the intertubular fluid flow reaches up to the surface and endangers the resin - dentin bond strength . more scanning electron microscopy studies are required to investigate the effect of temperature changes on resin - dentin interface . the sbs of studied etch- and -rinse system was increased by higher temperature of the washing water . however , the bond strength of clearfil se bond as a self - etching system did not change using different water temperatures . the authors of this manuscript declare that they have no conflicts of interest , real or perceived , financial or non - financial in this article . the authors of this manuscript declare that they have no conflicts of interest , real or perceived , financial or non - financial in this article .

background : the purpose of this study was to evaluate the effect of washing water temperature on the micro - shear bond strength ( sbs ) of composite resin to dentin using a two - step etch - and - rinse system and a two - step self - etching system.materials and methods : in this in vitro study , the intact dentins of buccal and lingual surfaces of healthy third molars were exposed . dentin surfaces were rinsed with different temperatures of distilled water ( 20 s ) before applying single bond ( sb ) or clearfil se bond(se ) . after applying the adhesive , composite cylinders ( 0.8 mm diameter and 1 mm length ) were bonded to the teeth surfaces . after storing the specimens in 37c distilled water for 48 h and thermocycling , sbs test was done . data were analyzed using analysis of variance , post hoc tukey tests , paired samples t - test , and fisher exact test ( = 0.05).results : temperature and interaction of temperature and type of bonding agent affected the bond strength . the bond strength of sb groups was significantly higher at 50c washing than 5c ( p = 0.003 ) and 22c ( p = 0.019 ) , but no significant difference was observed between se groups . the bond strength of se was significantly higher at 22c than that of sb ( p = 0.031 ) , whereas the bond strength of sb was significantly higher at 50c than that of se ( p = 0.007).conclusion : the use of high - temperature washing water is an appropriate method to enhance bond strength in etch - and - rinse systems .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

PMC4848073

it is well recognized that mobility is an important factor for the social participation and quality of life of individuals . for people who live with locomotor impairments , mobility assistive devices such as manual wheelchairs , power wheelchairs ( pw ) , scooters , and other motorized vehicles can be facilitators of mobility . the increasing prevalence of chronic disease and the changing demographics of our society will increase the use of mobility assistive devices such as pws by older adults . using a pw is a complex task whose efficiency and safety are modulated by factors such as individual capacity , wheelchair driving skills , design and technology features of the pw , environmental considerations , and interaction effects between these factors . although pw mobility has many potential benefits for users , pw incidents and accidents are not uncommon and their consequences can be serious . users of pws can have difficulty maintaining a supported seated posture when subjected to external forces , . oscillatory and shock whole body vibration ( wbv ) can be induced by ordinary obstacles or surfaces that pw users encounter every day and are often linked to speed control of the pw . these incidents can be associated with unsafe behaviours combined with poor pw driving skills , . pw driving skills are typically modulated by experience and joystick control but can be improved through training , . few objective outcome measures used in the clinical assessment of pw mobility take into account all the factors influencing pw mobility and safety . pw driving skills are typically evaluated with performance - based measures during standardized driving tasks in controlled or real environments , and graded by an observer who will pass judgement on the individual s skill level . insufficient research has been published on the ability of these instruments to extrapolate to pw mobility and safety in everyday use . indeed , while pw driving skills are an important factor in the safe operation of a pw , user behaviour and risk mitigation by the user during everyday use of the pw is as important . in order to educate pw users on risk mitigation and improve their powered wheelchair driving skills , a better characterization of pw users driving behaviour is required , in various real and ecological indoor and outdoor settings . ambulatory real life monitoring approaches of pw use with embedded sensors and datalogging are starting to emerge . cooper et al . determined the driving characteristics of pw users during unrestricted community activities by using a sensor attached to a pw and a custom - built datalogger . speed , distance traveled , and the time that each subject s personal wheelchair was being driven were recorded for 24hr / d over approximately five days for each subject . drivers of pw were most active during the afternoon and evening hours . over the 5-day period of this study , there was little variation in the speed or distance driven per day across subjects with an average daily distance of 1.7 km . reported pw usage using instrumentation known as the wheelchair activity monitoring instrument ( whami ) . pw usage was logged electronically , and geo - location and interview data were used to attribute pw use to ( 1 ) in the home , ( 2 ) not in the home indoors , or ( 3 ) outdoors , over a sample of twenty - five non - ambulatory , full - time pw users . distance wheeled , time spent wheeling , number of bouts , time spent in the wheelchair , and the percentage of time spent wheeling were measured to describe wheelchair use . overall , most research into pw mobility using embedded sensors and datalogging have reported global metrics on use of pw , such as the total distance and time spent moving each day , and have not taken into account context specific information on pw . to improve our understanding of pw use , its potential interrelationship with the environment and the driving skills of users , and ultimately its impact on the mobility of older adults , better outcome measures of pw mobility need to be developed . advances in data mining methods combined with the availability of low cost sensing technologies provide new opportunities to automatically classify pw activity , and understand behaviours of pw users in natural environments . in our previous work , we presented a method to automatically recognize and categorize five different driving activities during pw use , directly from sensor data . the method trains a support vector machine classifier , using time - delay embedding features of sensor - based data collected from a datalogging platform installed on a pw . the objectives of the present study are to : 1 ) test the robustness of the events recognition method with a dataset containing a larger spectrum of pw activities ( 35 instead of 5 ) , 2 ) assess its performance to automatically classify safe vs. unsafe activities using a very limited training set ( only five examples of each activity ) . fig . 1 provides an overview of the multiple components of our end - to - end system for automatic detection and classification of pw driving activities . first , the data - logging device handles real - time recording of sensor data onboard the pw . second , event segmentation is applied to the recorded data to identify events of interest from the data - stream . next , feature extraction computes a rich set of features from the events of interest . finally , we apply the machine learning classifier to categorize the events according to type ( safe vs. unsafe ) and activity ( 35 different classes ) . figure 1.classification process using different features . a proof of concept for a measurement approach to monitor real life use of pw driving was developed and is currently in use with a sample of pw users . the platform , called the wireless inertial measurement unit with gps ( wimu - gps ) ( fig . 2 ) , consists of a datalogger with embedded sensors and external sensors installed on the pw . the wimu - gps contains a number of sensors and components . in this paper the rationale for this choice is to limit the number of sensor inputs in order to explore the power of the proposed event detection and classification methods under a set of distinct and similar driving tasks . accelerometers are inexpensive , reliable , and unobtrusive , thus would be easy to install on any pw . in our case , the wimu - gps is installed on the base near the wheel , which takes only a few minutes and minimal tools to install . once the datalogging platform has been installed on the pw ( an oasis model from orthofab ) , a wheelchair user ( in our case a research assistant ) is asked to perform 35 distinct activities , listed in table 1 . the activities are selected so that they cover most common wheelchair activities in different speeds , directions , and other conditions , following consultation with clinicians . of the 35 activities , 15 are identified as being safe activities , and 20 are identified as unsafe a priori . unsafe activities are defined as those activities where collisions with objects occurred at different speeds . the remaining activities are defined as the safe activities . the rational for the choice and segmentation of the driving tasks ( safe or unsafe ) is based on the assumptions that irrespective of the task performed , impacts ( collisions ) are detrimental to the safety of the user as they have difficulty maintaining a supported seated posture when subjected to external forces . as such , while manoeuvring an pw , collisions can occur from different point of the pw ( forward motion , backward motion , side motion ) at different speeds . many of the safe activities selected ( such as passing a step at high speed ) can potentially be mistaken with the unsafe activities . this is done intentionally to ensure that our event classification process leads to robust classification of activities . each activity is repeated 30 times ( each of these repetitions is considered an event ) . the 30 events for each activity are then concatenated to form a quasi - periodic signal for each activity . the 30 events are further separated into six cross - validation sets , each containing five repetitions of each activity . the reason for this is two - fold : it allows us to show that the system can be trained from relatively few examples ( five events for each activity ) , and by having cross - validation sets we can get a confidence estimate on our results . the goal of this component is to extract events - of - interest from the input signal ( without , at this stage , identifying the activity s type ) . segmentation of the input signal into events - of - interest during long - term recordings is an essential processing step to make the proposed approach viable . if we were to keep all the data for the classification steps , then we would face a substantial class imbalance problem , whereby the activity corresponding to standard driving ( without an event - of - interest ) would constitute a much larger portion of the dataset than the specific activities . this often makes the classification task unnecessarily difficult for the algorithms . to segment the acquired data , accelerometer signals \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s = $ \end{document } ( x , y , z ) are partitioned into non - overlapping windows of length \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ l$ \end{document } , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ l$ \end{document } is the average duration of one event . for example , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ ( x_{t},x_{t+1 } , \ldots , x_{t+l } ) $ \end{document } forms the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ i$ \end{document}th window s \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ x$ \end{document } coordinate , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ i=\frac { t-1}{l}+1 $ \end{document}. then for each data point \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{t}=(x_{t } , y_{t } , z_{t } ) $ \end{document } in window \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { w}_{i}$ \end{document } , the normalized acceleration magnitude , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta _ { t}$ \end{document } , is computed for each data point:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } \beta _ { t } = \sqrt { \left ( { \frac { x_{t } -\mu _ { xi } } { \sigma _ { xi } } } \right ) ^{2}+\left ( { \frac { y_{t } -\mu _ { yi } } { \sigma _ { yi } } } \right ) ^{2}+\left ( { \frac { z_{t } -\mu _ { zi } } { \sigma _ { zi } } } \right ) ^{2 } } \end{equation*}\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu _ { si , s \in \{x , y , z\}}$ \end{document } indicates the mean of over time window \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { w}_{i}$ \end{document}.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } \mu _ { is}=\frac { \sum \limits _ { t\in { w}_{i } } s_{t } } { l } , s\in \ { x , y , z \ } \end{equation*}\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \sigma _ { is}^{2 } , _ { s \in \{x , y , z\}}$ \end{document } indicates the variance of over the same window . in this case , variance is defined by the squared deviation of each signal value from its expected value or mean.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } \sigma _ { is}^{2}=\frac { \sum \limits _ { t\in { w}_{i } } ( s_{t}-\mu _ { is } ) ^{2 } } { l } , s\in \ { x , y , z \}. \end{equation*}\end{document } afterwards , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta ( .)$ \end{document } is smoothed by the autoconvolution function . after the preprocessing step , accelerometer data points in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s$ \end{document } are clustered into two groups to differentiate between events - of - interest and baseline activity based on the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta ( .)$ \end{document } signal . clustering is achieved using the k - means clustering algorithm , which partitions data into \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ k$ \end{document } disjoints subsets such that each data point belongs to the cluster with the nearest centroid ( we assume \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ k=2 $ \end{document } ) . one cluster is composed of events - of - interest , to be further classified , and all the subsequent steps of our methodology are applied only to the data points that fall in this cluster . one of the core components of the system is the extraction of appropriate features on which to perform automatic classification . we consider four different types of features , each of which focuses on different signal properties : time - delay embedding features , simple time - domain features , frequency - domain features , and wavelet transforms . one of the contributions of this paper is to provide a detailed comparison of their performance on a challenging dataset . the first feature set considered contains simple characteristics of the time - series : mean , variance and line length on each of the three accelerometer coordinates . these time - domain signal properties are simple to measure and have shown good results in classification tasks of time - series . the mean , variance and line length features are generated for windows , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { w}_{i}$ \end{document } of size \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ l$ \end{document } from the segmented data . therefore , the feature vector for the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ i$ \end{document}th window is { \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu _ { xi},\mu _ { yi},\mu _ { zi},\sigma _ { xi}^{2},\sigma _ { yi}^{2},\sigma _ { zi}^{2 } , ll_{xi } , ll_{yi},ll_{zi}$ \end{document } } , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mu $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \sigma ^{2}$ \end{document } are defined as in the previous section . the line length of a signal in a time window indicates the sum of absolute changes in the signal amplitude during that period : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } ll_{is}=\sum \limits _ { t\in { w}_{i } } { \left | s_{t}-s_{t-1 } \right | , s\in \left \ { x , y , z \right \}. } \end{equation*}\end{document } the second set of features considered is the magnitude of fft coefficients for each of the accelerometer dimensions , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ x , y , z$ \end{document}. although it is more common to use the power spectrum of fft coefficients , different trials showed that the magnitude of coefficients has better accuracy for classification of dynamic activities . using a discrete fourier transform ( dft ) , we extract \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \{{f}_{ix}^{1 } , f_{ix}^{2 } , \ldots , f_{ix}^{n } , f_{iy}^{1 } , \ldots , f_{iy}^{n } , f_{iz}^{1 } , \ldots , f_{iz}^{n}\}$ \end{document } , the fft feature vector corresponding to window \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { w}_{i } $ \end{document } of the signal containing \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ l$ \end{document } data points , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ f_{is}^{k}$ \end{document } indicates the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ k$ \end{document}th fft coefficient of signal \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s$ \end{document}:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } f_{is}^{k}=\sum \limits _ { t\in { w}_{i } } { s_{t}e^{\frac { 2\pi jk}{l}t } } , s\in \{x , y , z\}. \end{equation*}\end{document } usually , only the first \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ n$ \end{document } coefficients for each signal are computed , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ n$ \end{document } is chosen based on time constraints and desired classification accuracy ; we use \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ n=10 $ \end{document } throughout our analysis . in addition to fft coefficients , a vector of wavelet transform coefficients is also considered . using wavelet analysis , the original signal is decomposed into coefficients containing both temporal and spectral information . this information can be used to identify the point at which the activity type is changed . studies have shown that wavelet coefficients extracted from accelerometer signals can provide useful information for activity analysis , . the decomposition is as follows:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{align } c ( n ) = & h_{0}s ( 2n ) + h_{1}s ( 2n+1 ) + h_{2}s ( 2n+2 ) + h_{3}s(2n+3)\notag \\ \\ d ( n ) = & h_{0}s ( 2n ) -h_{1}s ( 2n+1)+h_{2}s ( 2n+2 ) -h_{3}s(2n+3)\notag \\ { } \end{align } \end{document } where the multipliers are:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{align } h_{0}=\frac { 1+\sqrt { 3 } } { 4\sqrt { 2 } } , h_{1}=\frac { 3+\sqrt { 3 } } { 4\sqrt { 2 } } , h_{2}=\frac { 3-\sqrt { 3 } } { 4\sqrt { 2 } } , h_{3}=\frac { 1-\sqrt { 3 } } { 4\sqrt { 2 } } \notag \!\!\!\!\\ { } \end{align } \end{document } in our analysis , we consider the magnitude of the daubechies 2 wavelets for the 3d accelerometer data , as recommended in previous work . therefore , the corresponding feature vector for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathrm { w}_{\mathrm { i}}$ \end{document } with length \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ l$ \end{document } is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \{{\vert d}_{ix}^{1}\big | , { \vert d}_{ix}^{2 } \big | , \ldots , { \vert d}_{ix}^{n}\big | , { \vert d}_{iy}^{1 } \big |,\ldots , { \vert d}_{iy}^{n}\big | , { \vert d}_{iz}^{1 } \big | , \ldots , \big | d_{iz}^{n } \big |\}$ \end{document } , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ d_{is}^{k}$ \end{document } indicates the detail coefficient at the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ k$ \end{document}th level of decomposition of signal \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s\mathrm { \in } \ { x , y , z \}$ \end{document } in time window \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ { w}_{i}$ \end{document}. a proper value for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ n$ \end{document } can be chosen based on time constraints and desired classification accuracy . the final set of features considered is based on a time - delay embedding of the accelerometer signal . the goal of this method is to characterize unknown dynamical systems directly from sampled data , . the time - delay embedding projects the dynamic properties of the time - series at time \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ t$ \end{document } in an m dimensional space \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \tilde { s_{t}}^{e}=(s_{t},s_{t - t } , \ldots s_{t - mt})$ \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ t\mathrm { > 1}$ \end{document } is the sampling delay and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ m\mathrm { > 1}$ \end{document } is an integer multiplying the sampling delay . the components of this state space form a coordinate system that captures the structure of the time - series . theoretically , we know that any time - delay structure can reconstruct the dynamics of the system if \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ m$ \end{document } is large enough and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ t $ \end{document } does not have a conflict with any periodic property of the system . but in practice , it remains a challenge to find the real dimension \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ d$ \end{document } and determine the proper sampling delay \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ m$ \end{document } necessary to capture the dynamics of the system . in general , statistical tools are used to find the parameters and the optimised values are used for further analysis . alternately , and as we do in this paper , one starts with the intended analysis and optimizes the result for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ m$ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ t$ \end{document } using a grid search over parameter space on a small validation dataset . after generating the high dimensional model , principal component analysis is used to map the system to the lower dimensional space . in this method , each set of observations in the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ m$ \end{document}-dimensional space , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \tilde { s_{t}}^{e}\in r^{m}$ \end{document } , is mapped to a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ p$ \end{document}-dimensional space , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{t}^{e}\in r^{p}$ \end{document } , ( with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ p < m)$ \end{document } using an orthogonal transformation . after mapping all data points to the new \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ p$ \end{document}-dimensional space , the distance from each data point to its nearest neighbour in each ( labelled ) activity set is measured , and this measurement forms the time - delay embedding feature . in other words , if the dataset includes \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ u$ \end{document } different activities { \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ a_{1},\ldots , a_{u}$ \end{document } } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ s_{ti}^{e}$ \end{document } indicates the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ p$ \end{document } dimensional embedding for the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ i$ \end{document}th activity at time \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ t$ \end{document } , the corresponding feature vector is \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ f ( i , t ) = { \{\vert s_{ti}^{e}-s_{t^{'}j}^{e}\vert \}}_{j=1,\ldots , u}$ \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ t^{'}=argmin\vert s_{ti}^{e}-s_{t^{'}j}^{e}\vert \}$ \end{document}. these pairwise distances are used as features for the subsequent classification step . for points that are members of the labelled set , the pairwise distance to their second nearest neighbour is used.the projected points are stored using a kd - tree structure . this structure is useful for fast searching because it organizes the data points spatially , and can respond to pairwise distance queries in time linear in the number of points and in the dimension of the tree . this structure is useful for fast searching because it organizes the data points spatially , and can respond to pairwise distance queries in time linear in the number of points and in the dimension of the tree . for our experiments , we build three separate time - delay embeddings ( corresponding to recorded accelerometer data in the x , y , and z axes , respectively ) for each of the 35 activity types . the parameters are the same for all embeddings , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ m = 16 $ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ t = 0.1 $ \end{document } sec and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ p = 5 $ \end{document}. given a dataset where each point is described by a feature vector { \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ f\big ( x_{t}^{i } \big ) , f\big ( y_{t}^{i } \big ) , f\big ( z_{t}^{i } \big ) \}~i=1 , \ldots , n$ \end{document } , supervised learning techniques can be applied to find a mapping from feature space to event identity . for this step , an svm classifier constructs a set of hyperplanes in a high - dimensional space that classifies the samples into two classes . the classifier is essentially a function mapping the feature vector \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ f(x , y , z)$ \end{document } to the output set , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \mathrm { \gamma \in } ~\ { \mathrm { 1,-1 } \}.$ \end{document } to construct these hyperplanes , the svm uses an iterative training algorithm to minimize an error function , corresponding to the following optimization problem.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{align}&\hspace { -1.5pc}\min \nolimits _ { \mathrm { \omega , b , \zeta } } { \frac { 1}{2}\mathrm { \omega } ^{\mathrm { t}}\mathrm { \omega + c}\sum \nolimits _ { \mathrm { i=1}}^{\mathrm { n } } \mathrm { \zeta } _ { \mathrm { i } } } \notag \\&\hspace { -1.5pc}\mathrm { subject~ to } ~\mathrm { \gamma } _ { \mathrm { i } } ( \mathrm { \omega } ^{\mathrm { t}}\mathrm { \phi } ( \mathrm { f}_{\mathrm { i } } ) \mathrm { \,+ b } ) \mathrm ~{\ge \ , 1\,- } \mathrm { \zeta } _ { \mathrm { i}},~\mathrm { \zeta } _ { \mathrm { i}}\mathrm ~{\ge \ , 0 } \end{align } \end{document } the feature vector is mapped to a higher dimensional space by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \phi $ \end{document } and the classifier finds a separating hyperplane in this space . svm classifiers are characterized by their kernel function , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ k\big ( f_{i},f_{j } \big ) = \phi ( f_{i } ) ^{t}\phi ( f_{j})$ \end{document}. we use a basic polynomial kernel : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ k\big ( f_{i},f_{j } \big ) = \big ( { \alpha f}_{i}^{t}f_{j}+r \big ) ^{d } , \alpha > 0 $ \end{document } , with degree \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ d=2.$ \end{document } in order to classify a dataset including multiple classes , multiclass svm can be achieved by reducing a single classification problem into multiple binary classification problems . we begin by considering the segmentation of events - of - interest from the datastream . , we considered datapoints at the full time resolution ( 250 hz ) . the magnitude signal \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta $ \end{document } was generated over windows of size 7000 points ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ = 28 $ \end{document } sec ) , the average duration of each activity . then \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta $ \end{document } is smoothed by auto - convolving \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta _ { t}$ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta _ { t+}{_{\mathrm { \delta } } } $ \end{document } ( where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \delta = 100 $ \end{document } points @ 250hz ) . finally , the datapoints in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ \beta $ \end{document } were divided into two clusters using the k - means algorithm . we applied a final smoothing step over the clustering output , removing cluster assignments with fewer than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ k$ \end{document } consecutive points ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ k=1500 $ \end{document } points , selected by cross - validation ) . for most activities , we correctly segmented the 30 repetitions of each activity . 3a presents a sample segmentation , based on 30 repetitions of the dp1l ( rolling down 1 inch slope at low speed ) activity . for a few of the activities , some of the events were segmented into two parts . in particular , for three of the activities ( mp1l , mp1n , mp1r ) , each event - of - interest was segmented into two separate events , rather than a single one . 3b , where we observe that each event is characterized by a double - peak . for the other activity types ( excluding mp1l , mp1n , mp1r ) , out of 960 events , our approach missed one event ( 0.1% ) and over - counted 12 events ( 1% ) . figure 3.(a ) activity dp1l ( rolling down 1 inch slope at low speed ) , 30 repetitions , segmented into two groups : events - of - interest ( red boxes ) and background data ( blue signal ) . ( b ) activity mp1l ( climbing up 1 slope at low speed ) , 30 repetitions , segmented similarly ; in this case the automatic segmentation yielded 60 events - of - interest . ( a ) activity dp1l ( rolling down 1 inch slope at low speed ) , 30 repetitions , segmented into two groups : events - of - interest ( red boxes ) and background data ( blue signal ) . ( b ) activity mp1l ( climbing up 1 slope at low speed ) , 30 repetitions , segmented similarly ; in this case the automatic segmentation yielded 60 events - of - interest . the data points extracted via segmentation are further processed for feature extraction according to the four types of features outlined above . features of the time domain , fft coefficients , and wave transforms , were extracted using the automated feature extraction and selection toolbox ( afest ) . features for the time - delay embeddings were produced using our own code . using these features vectors , we considered two types of classification tasks : binary classification to distinguish between safe and unsafe events , and per - type classification whereby events are categorized into the 35 types listed in table 1 . in this section we present the results of the safe vs. unsafe classification task . the results of the per - type classification task are described in the following section . to train and evaluate the classifiers , the data corresponding to the events - of - interest were divided into two datasets : a training set that was used to train the algorithms , and included five trials ( out of 30 ) for each activity ; and a test set that was used to evaluate the performance of the algorithm , and included five different trials . we used a small training set as this is more realistic for eventual deployments where assessment of pw driving skills for a given pw user could include tasks needed to generate this training set . all reported results are the average of a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ k$ \end{document}-fold cross validation ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ k=6 $ \end{document } ) . feature extraction , with all types of features , was done over windows of 1.6 sec , assuming a 1.596 sec ( = one data point ) overlap with the preceding window . the average classification error to detect safe and unsafe activities using each type of features is shown in fig . we also examined the classification accuracy for each of the 35 different activity classes separately ; results are presented in fig . figure 4.average error rate ( % ) over all safe / unsafe events , using five different types of features : time - delay embeddings , time - based features , fft coefficient magnitudes , wavelet transform , and all features . error is defined as the number of false positives and false negatives over the total number of examples . figure 5.safe vs. unsafe classification average error rate ( % ) per activity class , using 5 different types of features : time - based features , fft coefficient magnitudes , time - delay embeddings , wavelet transforms , and all features . the average error rate for each event was calculated over six folds of cross - validation . in each fold , the training set included five examples of each activity . average error rate ( % ) over all safe / unsafe events , using five different types of features : time - delay embeddings , time - based features , fft coefficient magnitudes , wavelet transform , and all features . error is defined as the number of false positives and false negatives over the total number of examples . safe vs. unsafe classification average error rate ( % ) per activity class , using 5 different types of features : time - based features , fft coefficient magnitudes , time - delay embeddings , wavelet transforms , and all features . the average error rate for each event was calculated over six folds of cross - validation . in each fold , the training set included five examples of each activity . using only simple time - domain features , our classification algorithm incorrectly classified 9% of unsafe events as being safe , and 17% of safe events were classified as unsafe , as shown in fig . 5 that two out of 20 unsafe activities were classified as being safe more than 20% of the time . using only fft coefficients , results in fig . 4 and fig . 5 show that we can achieve a low classification error for unsafe events ( 3% ) . the fft features also achieved the lowest error ( 33% ) on the challenging ifmr ( frontal collision with a moving object at high speed ) activity . however the safe dp1l ( rolling down 1 inch slope at low speed ) activity was always misclassified as being unsafe . 4 , detection of events with time - delay embeddings misclassified 7% of unsafe events and 15% of safe events . the most difficult unsafe activity to classify was once again ifmr ( frontal collision with a moving object at high speed ) . of the safe activities , the tde features were the only ones to successfully classify dp1l ( rolling down 1 inch slope at low speed ) , with only 3% error . 5 that classification errors for unsafe activities were localized to certain activity types , for example ifcr ( frontal collision with a soft object at high speed ) and ifmr ( frontal collision with a moving object at high speed ) for the unsafe activities . finally , we considered the classification accuracy that can be achieved when combining all feature families within a single classifier . as shown in fig . 4 , with this combination , our classification algorithm achieved a misclassification rate of 2% for unsafe events and 12% for safe events . this was lower than with any single feature family , but not significantly better than with the fft features . this may be due to the small size of the training set ; in general , larger training sets are necessary when training classifiers with greater numbers of parameters ( features in this case ) . this classifier outperformed all the others on most activities , though there were a number of activities for which the time - delay embedding features or fft features performed better , notably ifcr ( frontal collision with a soft object at high speed ) . running paired t - tests comparing the misclassification rates obtained with the various feature sets confirmed that we could partition feature families into two sets : { fft features , all features } vs. { time - based features , wavelet features}. results obtained with any of the feature types in the first set were significantly better ( at the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } $ p=$ \end{document } 0.05 level ) than those obtained with any of the features in the second set , but not significantly better than those obtained within their set . there was no significance between time - delay embeddings and any of the other feature families . we end this section by presenting results showing the impact of the training set size on the safe / unsafe classification performance . we focus on the fft family of features and the allfeatures set , which performed best in the results above . that the safe / unsafe classification performance improved steadily until the 5 first training examples , and continued to improve but at a slower rate , up to 15 training examples for fft features . in the case of the allfeatures , improvements continued up to 25 training examples ( the limit of our dataset , preserving a five example test ) and may improve further with more data . this confirms that moderate amounts of data can be sufficient to correctly classify events , but that more data can be useful , especially when considering a greater number of features . the paper presents a machine learning approach targeting the development of automated analysis tools to characterize the driving behavior of wheelchair users . the results presented constitute a proof - of - concept that the system can accurately detect unsafe activities using a variety of features and support - vector machine classification , with less than 2% error , and a relatively low rate of false positives ( 12% ) . this approach can be helpful in monitoring safe usage of the wheelchair under varied operating conditions . the number of false alarms can likely be reduced by leveraging richer sensor data ( e.g. , using audio or video data to better diagnose activities ) . offering more training examples in a diversity of safe activities we compared classification performance using different sets of features common in the time - series analysis literature : time - delay embeddings , time - domain characterization , frequency - domain features , and wavelet transforms . this was deemed necessary because these features have been often used in the literature , without reliable comparison between them . the results presented here show that the most robust performance was achieved when combining all features , however nearly as good performance was obtained by using fft features alone . our approach could be helpful in diagnosing frequent driving mistakes in every day usage , without requiring a user to keep a detailed diary . one of the challenges in moving forward with the implementation of this technique onboard regular pws is to validate the method using natural recording conditions . the labels could however be assigned through post - processing of the recorded data , and corroboration through camera data . furthermore , the current system was evaluated using activity recordings from a single user ; we expect results to be relatively robust to different users given the very narrow class of events considered , however this remains to be tested .

using a powered wheelchair ( pw ) is a complex task requiring advanced perceptual and motor control skills . unfortunately , pw incidents and accidents are not uncommon and their consequences can be serious . the objective of this paper is to develop technological tools that can be used to characterize a wheelchair user s driving behavior under various settings . in the experiments conducted , pws are outfitted with a datalogging platform that records , in real - time , the 3-d acceleration of the pw . data collection was conducted over 35 different activities , designed to capture a spectrum of pw driving events performed at different speeds ( collisions with fixed or moving objects , rolling on incline plane , and rolling across multiple types obstacles ) . the data was processed using time - series analysis and data mining techniques , to automatically detect and identify the different events . we compared the classification accuracy using four different types of time - series features : 1 ) time - delay embeddings ; 2 ) time - domain characterization ; 3 ) frequency - domain features ; and 4 ) wavelet transforms . in the analysis , we compared the classification accuracy obtained when distinguishing between safe and unsafe events during each of the 35 different activities . for the purposes of this study , unsafe events were defined as activities containing collisions against objects at different speed , and the remainder were defined as safe events . we were able to accurately detect 98% of unsafe events , with a low ( 12% ) false positive rate , using only five examples of each activity . this proof - of - concept study shows that the proposed approach has the potential of capturing , based on limited input from embedded sensors , contextual information on pw use , and of automatically characterizing a user s pw driving behavior .

Introduction Methods and Procedures Results Conclusion

PMC5338722

culturomics is a new approach introduced in our laboratory in 2012 that aims to study the diversity of the microorganisms colonizing the human gut . recently culturomics was applied to explore the bacterial diversity of patients with kwashiorkor . during this study , we isolated the strain mt9 (= csur p1511 = dsm 102954 ) , which belongs to a new bacterial genus within the family clostridiaceae . this family was created by ernst august pribram in 1933 and contains as its core the genus clostridium and 36 other genera . they have been isolated from highly diverse habitats , including freshwater and marine sediments , salt lakes , various sites of the human body , manure piles , sewage sludge and different types of faeces . so far , the criteria used to classify new species based on 16s rdna sequencing and dna - dna hybridization ( ddh ) , are limited due to the low cutoff between species and genus . recently we proposed a new polyphasic approach associating phenotypic characterization , matrix - assisted desorption ionization time of flight mass spectrometry ( maldi - tof ms ) , and genotypic characteristics to better define and classify new species . in addition , characterizing new species by sequencing the whole genome allows a better distinction between the closest related species , comparing the whole genome and all of its coded function , and genomic data to become available for new bacterial species . in this culturomics study , we isolated the strain mt9 (= csur p1511 = dsm 102954 ) , which is the first representative of a new bacterial genus within the family clostridiaceae . here the stool specimens were collected from a 1-year - old senegalese patient with kwashiorkor after defecation in sterile plastic containers . the stool specimen was then formed into aliquots and stored at 80c until use . the study and the assent procedure were approved by the national ethics committee of senegal and the local ethics committee of the ifr48 ( marseille , france ) under numbers 11 - 017 and 09 - 022 , respectively . the stool sample was cultivated in anaerobic conditions and preincubated in a blood culture bottle ( becton dickinson , le pont - de - claix , france ) at 37c for 15 days after a thermic shock at 80c for 20 minutes . then 1 ml of the enriched culture was spread on a 5% sheep 's blood enriched columbia agar ( biomrieux , marcy l'etoile , france ) and incubated at 37c for 72 hours . the obtained colonies were purified and identified using maldi - tof ms as previously described , . this proteomic analysis was conducted using a microflex spectrometer ( bruker daltonics , leipzig , germany ) with a mtp 96 maldi - tof target plate ( bruker ) . the obtained spectra were imported into maldi biotyper 2.0 software ( bruker ) and analysed by standard pattern matching ( with default parameter settings ) against the 7567 references contained in our database ( bruker database incremented with our data ) . when the identification score was 1.9 , the species level was determined ; a score between 1.9 and 1.7 allowed identification only at the genus level ; and a score < 1.7 gave no identification . in this last case , the 16s rrna gene was sequenced as previously described in order to get a molecular identification . stackebrandt and ebers determined a 98.7 and 95% similarity level threshold to define a new species and a new genus respectively without performing ddh . growth of the strain mt9 was tested under anaerobic and microaerophilic conditions generated using genbag anaer and genbag microaer systems ( biomrieux ) respectively , and in aerobic conditions with or without 5% co2 . optimal salt concentration was determined by growing the strain at 0 , 0.5 , 1 and 1.5% nacl . different ph conditions were tested : 6 , 6.5 , 7 , 7.5 , 8 and 8.5 . the biochemical characteristics of the strain mt9 were studied using api zym , api 20 ne and api 50 ch strips ( biomrieux ) according to the manufacturer 's instructions . clavulanic acid , clindamycin , metronidazole , vancomycin , doxycycline , imipenem , rifampicin and trimethoprim / sulfamethoxazole using antibiotic discs ( i2a , montpellier , france ) . the surface colonies of the strain mt9 was observed after 72-hour incubation under anaerobic conditions at 37c . in order to observe cell morphology , cells were fixed with 2.5% glutaraldehyde in 0.1 m cacodylate buffer for at least 1 hour at 4c . a drop of cell suspension was deposited for approximately 5 minutes on glow - discharged formvar carbon film on 400-mesh nickel grids ( fcf400-ni ; electron microscopy sciences ( ems ) , hatfield , pa , usa ) . the grids were dried on blotting paper , and cells were negatively stained for 10 seconds with 1% ammonium molybdate solution in filtered water at room temperature . electron micrographs were acquired with a tecnai g20 cryo ( fei company , limeil - brevannes , france ) transmission electron microscope operated at 200 kev . to determine the gram stain strain mt9 was observed under a dm1000 photonic microscope ( leica microsystems , nanterre , france ) after gram colouration as well as a fresh growing culture to assess its motility . two samples were prepared with approximately 15 mg of bacterial biomass per tube collected from several culture plates . briefly , fame were separated using an elite 5-ms column and monitored by ms ( clarus 500-sq 8 s , perkinelmer , courtaboeuf , france ) . spectral database search was performed using ms search 2.0 operated with the standard reference database 1a ( national institute of standards and technology ( nist ) , gaithersburg , usa ) and the fames mass spectral database ( wiley , chichester , uk ) . after a pretreatment by a lysozyme incubation at 37c for 2 hours , dna of strain mt9 was extracted on the ez1 biorobot ( qiagen , germantown , md , usa ) with ez1 dna tissues kit . genomic dna ( gdna ) was quantified by a qubit assay with the high sensitivity kit ( life technologies , carlsbad , ca , usa ) to 31.7 ng/l . gdna of strain mt9 was sequenced on the miseq technology ( illumina , san diego , ca , usa ) with the mate pair strategy . the gdna was barcoded in order to be mixed with 11 other projects with the nextera mate pair sample prep kit ( illumina ) . the mate pair library was prepared with 1.5 g of gdna using the nextera mate pair illumina guide . the pattern of the fragmentation was validated on an agilent 2100 bioanalyzer ( agilent technologies , santa clara , ca , usa ) with a dna 7500 labchip . the dna fragments ranged in size from 1.5 to 11 kb , with an optimal size at 7.670 kb . no size selection was performed , and 600 ng of tagmented fragments were circularized . the circularized dna was mechanically sheared to small fragments with an optimal at 804 bp on a covaris device s2 in t6 tubes ( covaris , woburn , ma , usa ) . the library profile was visualized on a high sensitivity bioanalyzer labchip ( agilent technologies ) , and the final concentration library was measured at 9.39 after a denaturation step and dilution at 15 pm , the pool of libraries was loaded onto the reagent cartridge and then onto the instrument along with the flow cell . automated cluster generation and sequencing run were performed in a single 39-hour run in a 2 251 bp read length . total information of 6.1 gb was obtained from a 653k / mm cluster density with a cluster passing quality control filters of 96.1% ( 12 open reading frames ( orfs ) were predicted using prodigal with default parameters , but the predicted orfs were excluded if they were spanning a sequencing gap region . the predicted bacterial protein sequences were searched against the genbank database and the clusters of orthologous groups ( cogs ) database using blastp . the trnascanse tool was used to find trna genes , whereas ribosomal rnas were found by using rnammer and blastn against the genbank database . lipoprotein signal peptides and the number of transmembrane helices were predicted using signalp and tmhmm respectively . orfans were identified if their blastp e value was lower than 1e for alignment length greater than 80 amino acids . if alignment lengths were smaller than 80 amino acids , we used an e value of 1e . we compared the genome of strain mt9 to those of other members of the order clostridiales . their genomes were automatically retrieved from the 16s rrna tree using xegen software ( phylopattern ) . for each selected genome , the complete genome sequence , proteome genome sequence and orfeome genome sequence were retrieved from the ftp of national center for biotechnology information . this score is the mean value of nucleotide similarity between all couples of orthologues between the two genomes studied ( average genomic identity of orthologous gene sequences ; agios ) . an annotation of the entire proteome was performed to define the distribution of functional classes of predicted genes according to the clusters of orthologous groups of proteins ( using the same method as for the genome annotation ) . to evaluate the genomic similarity between anaeromassilibacillus genus and closely related genera , we determined two parameters : digital dna - dna hybridization ( dddh ) , which exhibits a high correlation with ddh , , and agios , which was designed to be independent from ddh . annotation and comparison processes were performed in the multi - agent software system dagobah that included figenix libraries to provide pipeline analysis . strain mt9 ( table 1 ) was isolated in january 2015 by anaerobic cultivation at 37c on 5% sheep 's blood maldi - tof ms results showed that the score obtained was under 1.7 for the strain mt9 , thus suggesting that our isolate was not a member of a known species listed in our database . 16s rrna gene sequencing and comparison demonstrate that the strain mt9 ( ln866991 ) exhibited 93.4% nucleotide sequence similarity with clostridium leptum ( nr114789 ) ( fig . 1 ) . this value was lower than the 95% 16s rrna gene sequence threshold recommended by stackebrandt and ebers to delineate a new genus without carrying out ddh . we incremented our database with the spectrum from strain mt9 ( fig . 2 ) . colonies of the strain mt9 were 1 to 2 mm in diameter on columbia agar , and opaque white in colour . motile and spore - forming , 2.40 m in length and 0.9 m in diameter ( fig . 5 ) . strain mt9 grows in a range from 25 to 37c , with an optimum at 37c . nacl is not required for growth , but strain mt9 was only able to grow between 0 and 0.5% nacl . using the api zym gallery , positive reactions were observed for alkaline phosphatase , esterase , esterase lipase , valine arylamidase , acid phosphatase , naphtol - as - bi - phosphohydrolase , -galactosidase , -galactosidase , -glucosidase , -glucosidase , n - acetyl--glucosaminidase and -mannosidase . the api 50ch gallery showed positive reactions for d - ribose , d - adonitol , dulcitol , d - sorbitol , amygdalin , arbutin , esculin , d - cellobiose , d - maltose , inulin , d - raffinose , amidone , glycogen , xylitol , gentiobiose , d - tagatose and potassium 5-ketogluconate . using the api 20 ne , enzymatic activities were observed for gelatin hydrolysis and -galactosidase . antibiotic susceptibility test demonstrated that strain mt9 is susceptible to vancomycin , doxycycline , ceftriaxone , imipenem , amoxicillin clavulanic acid , penicillin and rifampicin , and is resistant to trimethoprim / sulfamethoxazole , ciprofloxacin , oxacillin , cefoxitin , colistin and fosfomycin . the phenotypic and biochemical features of the strain mt9 were compared to the features of closely related species ( table 2 ) . the major fatty acids were hexadecanoic acid ( 32% ) , tetradecanoic acid ( 27% ) and 9-octadecenoic acid ( 26% ) . minor amounts of unsaturated , branched and other saturated fatty acids were also detected ( table 3 ) . the 3 511 289 bp long genome contains 52.94% of g+c content ( fig . 6 and table 4 ) and is composed of 12 scaffolds ( composed of 15 contigs ) . of the 3095 predicted genes , 3046 were protein - coding genes and 49 were rnas ( two genes are 5s rrna , one gene is 16s rrna , one gene is 23s rrna and 45 genes are trna genes ) . a total of 1773 genes ( 58.21% ) were assigned as putative function ( by cogs or by nr blast ) . the genomic characteristics of strain mt9 were compared to closely related species ( table 6 ) . the draft genome sequence of strain mt9 ( 3.51 mb ) is smaller than those of oscillibacter valericigenes , clostridium cellulolyticum , flavonifractor plautii , acetivibrio cellulolyticus , clostridium clariflavum and clostridium papyrosolvens ( 4.47 , 4.07 , 3.81 , 6.15 , 4.90 and 4.40 mb respectively ) , but larger than those of ethanoligenens harbinense , faecalibacterium prausnitzii and clostridium leptum ( 3.01 , 3.32 and 2.82 mb respectively ) . the g+c content of strain mt9 ( 52.94% ) is smaller than those of o. valericigenes , e. harbinense , f. prausnitzii and f. plautii ( 53.19 , 55.56 , 56.82 and 61.07% respectively ) , but larger than those of c. cellulolyticum , c. leptum , a. cellulolyticus , c. clariflavum and c. papyrosolvens ( 37.40 , 50.25 , 35.55 , 35.72 and 37.13% respectively ) . the gene content of strain mt9 ( 3046 ) is smaller than those of o. valericigenes , c. cellulolyticum , f. plautii , a. cellulolyticus , c. clariflavum and c. papyrosolvens ( 4723 , 3390 , 4278 , 5082 , 3892 and 3867 respectively ) , but larger than those of e. harbinense , f. prausnitzii and c. leptum ( 2701 , 2756 and 2482 respectively ) . strain mt9 shared respectively 866 , 787 , 841 , 789 , 766 , 818 , 681 , 780 and 772 orthologous genes with f. plautii , a. cellulolyticus , c. leptum , c. papyrosolvens , e. harbinense , o. valericigenes , f. prausnitzii , c. cellulolyticum and c. clariflavum . the comparison of the strain mt9 to other species resulted in agios values ranging from 57.07% with a. cellulolyticus to 67.67% with c. leptum ( table 7 ) . dddh was estimated between strain mt9 's genome and the other compared genomes , and results ranged from 19.6% with f. plautii to 30.4% with c. clariflavum ( table 8) . these values are in the range of the dddh values of compared species except our strain ( 18.832.2% ) and under the 70% threshold used to delineate a new species , thus confirming the status of the strain mt9 as a previously unknown member of the clostridiaceae family . the distribution of genes into cogs categories was similar , but not identical , in all compared genomes ( fig . for instance , o. valericigenes presented more genes dedicated to the dna replication , recombination and repair . the new polyphasic approach based on phenotypic , phylogenetic and genomic analyses ( taxonogenomics ) represented here support the affiliation of the strain mt9 to a new bacterial genus . , sp . nov . , is the first representative of this new genus for which the strain mt9 is the type strain . this strain was isolated from the stool specimen of a 1-year - old patient with kwashiorkor living in senegal . n. , anaero , from anar , the greek term for absence of oxygen , massili for massilia , the latin name of marseille , and bacillus , little rod ) . cells are gram - negative , motile , strictly anaerobic , spore - forming , rod - shaped bacilli . adj . , senegalensis , originating from senegal, the country from which the stool sample was collected ) . cells are gram - negative , motile , spore - forming , rod - shaped bacilli with a length of 2.4 m and a diameter of 0.9 m . colonies were 1 to 2 mm in diameter on columbia agar , and opaque white in colour . a. senegalensis strain mt9 is strictly anaerobic , grows at 37c ( optimum ) and ph 7 ( optimum ) and nacl is not required . positive reactions were observed for alkaline phosphatase , esterase , esterase lipase , valine arylamidase , acid phosphatase , naphtol - as - bi - phosphohydrolase , galactosidase , -galactosidase , -glucosidase , -glucosidase , n - acetyl--glucosaminidase , -mannosidase , d - ribose , d - adonitol , dulcitol , d - sorbitol , amygdalin , arbutin , esculin , d - cellobiose , d - maltose , inulin , d - raffinose , amidone , glycogen , xylitol , gentiobiose , d - tagatose and potassium 5-ketogluconate . antibiotic susceptibility test showed that a. senegalensis strain mt9 is susceptible to vancomycin , doxycycline , ceftriaxone , imipenem , amoxicillin clavulanic acid , penicillin , and rifampicin , and resistant to trimethoprim / sulfamethoxazole , ciprofloxacin , oxacillin , cefoxitin , colistin and fosfomycin . the 3 511 289 bp long genome exhibits a g+c content of 52.94% . the 16s rrna and genome sequences are deposited in embl - ebi under accession numbers ln866991 and cwip00000000 , respectively . the type strain mt9 (= csur p1511 = dsm 102954 ) was isolated from the gut microbiota of a 1-year - old senegalese patient with kwashiorkor .

using the culturomics strategy , a wide range of anaerobic bacteria was discovered including anaeromassilibacillus senegalensis strain mt9 t (= csur p1511 = dsm 102954 ) , isolated from the gut microbiota of a 1-year - old senegalese patient with kwashiorkor . this gram - negative strain is a strictly anaerobic , spore - forming rod motile by a polar flagellum . the 3 511 289 bp long genome of this strain contains 3046 protein - coding and 49 rna genes , including 45 trna and four rrna genes , and exhibits a g+c content of 52.94% . here we describe the features of this organism , together with the complete genome sequence and annotation .

Introduction Materials and Methods Results Conclusion

PMC3139068

migraine and epilepsy are both common neurological disorders and can occur as comorbid conditions [ 17 ] . from a genetic point of view , migraine is a polygenic multifactorial disorder , while many epilepsy syndromes are monogenic and have been pathogenetically linked to channelopathies . similarly familial hemiplegic migraine ( fhm ) , the only known autosomal subtype of migraine , is due to mutations in genes encoding ion channels , and it is often associated to epileptic seizures . previous studies have excluded the role of the two fhm genes ( cacn1a , atp1a2 ) in the common forms of migraine [ 8 , 1113 ] . considering the multifactorial etiology of migraine , genetic studies on phenotypically homogenous populations are required . the comorbidity between two conditions that share common pathogenetic mechanisms , such as migraine and epilepsy , could address the issue . the scn1a gene encodes the -subunit of the neuronal voltage - gated sodium channel nav1.1 ( mim # 182389 ) . mutations in scn1a are associated with a spectrum of epilepsy syndromes ; a few mutations result in fhm . so far , scn1a gene has never been investigated in subjects with common forms of migraine . the purpose of this study was to evaluate the role of the scn1a gene in the pathogenesis of comorbidity between primary headache and idiopathic or cryptogenic epilepsy in a large series of children and adolescents . this was a case control study comparing the genotype distribution and allele frequency of single nucleotide polymorphisms ( snps ) of scn1a gene between patients and healthy controls . fifty - six subjects out of 1,795 headache sufferers under the age of 18 consecutively diagnosed at the juvenile headache center of the department of pediatrics of padua hospital between 1995 and 2008 were selected according to the following criteria : ( 1 ) primary headache and ( 2 ) idiopathic or cryptogenic epilepsy or unprovoked seizures . patients with secondary headache or symptomatic epilepsy were excluded from the study . the methodology of patients recruitment and phenotype selection has been previously reported . the study protocol and consent / assent forms were approved by our institutional review board . all the healthy controls were interviewed by a board certified child neurologist regarding , in particular , the personal history of headache ; those affected by migraine or probable migraine , according to the criteria of the international classification of headache disorders ( ichd - ii ) were excluded . the molecular analysis was conducted at the genetic laboratory of the department of pediatrics ( university of padua ) . parental informed consent for genetic testing was obtained for each subject included in the study . genomic dna was extracted from peripheral blood using the high pure pcr template preparation kit ( roche diagnostics gmbh , mannheim , germany ) . genotypes were determined by high resolution melt ( hrm ) analysis [ 1719].table 1description of the polymorphisms of the scn1a gene in this studybase pair changeaminoacidpositionfrequency of heterozygous reported in literatureclinical associationreferencec.1748a > tp.d583 vexon 11ndnkncbic.2656 t > cp.s886pexon 15ndnkncbic.3199a > gp.t1067aexon 160.33the snp is more frequent in patients with epilepsy than in controls ( 33)[24 , 25 , 2733]c.5771 g > cp.i1955texon 260.025nknd not determined , nk not known description of the polymorphisms of the scn1a gene in this study nd not determined , nk not known the polymerase chain reaction ( pcr ) cycling and hrm analysis were carried out sequentially on a rotor gene 6000 ( corbett research , mortlake , australia ) using takara ex taq r - pcr custom ( takara bio europe sas , saint - germain - en - laye france ) . the hrm analysis was performed with the temperature ramping ( 70 to 95c , rising by 0.1c ) and fluorescence acquisition setting recommended by the manufacturer . the melting curves were normalized through calculation of the line of best fit of two normalization regions before and after the major fluorescence drop , corresponding to the melting of the pcr product using the software provided with the rotor gene 6000 ( corbett research ) . melting profiles and hrm running conditions were determined experimentally in 10 reference controls that were sequenced . we obtained three different melting profiles only for the 3199a > g polymorphism . we sequenced two samples for each melting profile of each polymorphism , confirming that different profiles corresponded to different sequences . sequence analysis revealed that heterozygotes and homozygotes have characteristic melting profiles that give rise to differently shaped melting curves . therefore , in the following hrm analyses we used the curves of homozygotes and heterozygotes that were confirmed by sequencing as the reference for the genotype analysis of the unknown samples . direct sequencing of pcr products with abnormal hrm profiles was performed with the use of the bigdye terminator v1.1 cycle sequencing kit ( applied biosystems ) on abi 310 automated dna sequencer ( pe applied biosystems , foster city , ca , usa ) . the hrm profiles of cases with comorbidity were compared to hrm profiles of healthy reference controls that were sequenced . genotype distributions in patients and controls were tested for hardy weinberg equilibrium , and differences in genotype frequencies between patients and controls were tested using a chi - square test for independence , with a level of significance set at 0.05 . among 1,795 headache sufferers under the age of 18 consecutively diagnosed at the juvenile headache center of the department of pediatrics of padua hospital between 1995 and 2008 , fifty - six subjects having comorbidity between primary headache and idiopathic or cryptogenic epilepsy or unprovoked seizures were selected . the clinical characteristics and the statistical analysis of these two population study have been previously reported . the present study focuses on the results of the scn1a gene molecular analysis , which was undertaken in 49 cases and 100 healthy controls . among the 49 cases , 45 subjects ( 92% ) had migraine ( m ) ( 38 migraine without aura , 7 migraine with aura ) and 4 patients suffered from episodic tension type ( etth ) headache . considering the type of epilepsy , there were 30 subjects with focal cryptogenic epilepsy ( 27 m , 3 etth ) , 11 with focal idiopathic epilepsy ( 10 m , 1 etth ) , and 8 ( 8 m , 0 etth ) with idiopathic generalized epilepsy . the hrm profiles consist of three different graphs ( melt temperature , normalized graph and the difference graph ) that are visualized all together during the analysis of results . as an example , we report the three graphs concerning the hrm analysis of exon 16 ( fig . 1 ) and of exon 11 ( fig . 2).fig . scn1a 3199a > g polymorphism ; green , homozygous for the a allele ( aa ) ; red , homozygous for the g allele ( gg ) ; blue , heterozygous ( ag)fig . 2rotor gene 6000 hrm normalized graph of the region amplified for exon 11 . scn1a 1748a > t polymorphism ; red ( sequenced reference controls ) , homozygous for the a allele ( aa ) ; green ( samples examined ) , homozygous for the a allele ( aa ) rotor gene 6000 hrm normalized graph of the region amplified for exon 16 . scn1a 3199a > g polymorphism ; green , homozygous for the a allele ( aa ) ; red , homozygous for the g allele ( gg ) ; blue , heterozygous ( ag ) rotor gene 6000 hrm normalized graph of the region amplified for exon 11 . scn1a 1748a > t polymorphism ; red ( sequenced reference controls ) , homozygous for the a allele ( aa ) ; green ( samples examined ) , homozygous for the a allele ( aa ) comparing the hrm profiles of the cases and healthy control samples with those of the sequenced reference controls , we could identify the genotypes of the samples examined . the polymorphism 3199a > g , having a significant heterozygosity ( 0.33 ) , exhibited three hrm profiles consistent with the three genotypes ( aa , ga , gg ) , as confirmed by direct sequencing . the other four snps having a low or not known heterozygosity disclosed a unique hrm profile , consistent with the wild type genotype . during hrm analysis of exon 11 , we found in one patient a different profile . the pcr product was analyzed by direct sequencing that disclosed a missense variation sequence 1811 g > a that causes at protein level a replacement of arginine with histidine ( r604h ) . the patient is a 15-year - old boy with a history of childhood absence epilepsy ( since 36 ) suffering from episodic tension headache , since the age of 11 . the analysis was carried out in both the parents ; the mother was negative while the father , who was asymptomatic , carried the missense variation sequence 1811 g > a. during the analysis of healthy controls , we found in one case a missense variation sequence 5782c > g that causes at protein level a replacement of arginine with glycine ( r1928 g ) . the allele and genotype frequencies , respectively , absolute and percentage , of the five snps observed in 49 patients and 100 healthy nonmigrainous controls , tested by hrm , are presented in table 2.table 2allele and genotype frequencies , respectively absolute and percentage ( in brackets ) , of the five snps of the scn1a gene observed in 49 cases and 100 controls , tested by hrmsnps of the scn1a gene1748a > t2656 t > c3199a > t > ccases ( n = 49 ) allele frequenciesa98 ( 100%)t98 ( 100%)a65 ( 66.3%)g98 ( 100%)t98 ( 100%)t0 ( 0%)c0 ( 0%)g33 ( 33.7%)a0 ( 0%)c0 ( 0% ) genotype frequenciesa / a49 ( 100%)t / t49 ( 100%)a / a22 ( 44%)g / g49 ( 100%)t / t49 ( 100%)a / t0 ( 0%)t / c0 ( 0%)a / g21 ( 44.7%)g / a0 ( 0%)t / c0 ( 0%)t / tc / cg / ga / ac / c0 ( 0%)0 ( 0%)6 ( 11.3%)0 ( 0%)0 ( 0%)controls ( n = 100 ) allele frequenciesa200 ( 100%)t200 ( 100%)a123 ( 61.5%)g200 ( 100%)t200 ( 100%)t0 ( 0%)c0 ( 0%)g77 ( 38.5%)a0 ( 0%)c0 ( 0% ) genotype frequenciesa / a100 ( 100%)t / t100 ( 100%)a / a42 ( 37.8%)g / g100 ( 100%)t / t100 ( 100%)a / t0 ( 0%)t / c0 ( 0%)a / g39 ( 47.4%)g / a0 ( 0%)t / c0 ( 0%)t / t0 ( 0%)c / c0 ( 0%)g / g19 ( 14.8%)a / a0 ( 0%)c / c0 ( 0% ) allele and genotype frequencies , respectively absolute and percentage ( in brackets ) , of the five snps of the scn1a gene observed in 49 cases and 100 controls , tested by hrm the analysis of the frequency distribution showed that , only for the polymorphism 3199a > g , the two alleles ( a : wild type ; g : mutated ) are represented both in cases and controls ; while for the other four snps the allele frequency of the wild type was 100% both in cases and controls , as assessed on a total of 298 alleles , therefore these snps are not true polymorphisms in the tested population . in the case of polymorphism 3199a > g , the genotype distributions for the two alleles ( a and g ) were in equilibrium under the hardy weinberg law both in cases ( p = 0.64 ) and controls ( p = 0.44 ) ( with chi - square test the balance is present if p > 0.05 ) . for this polymorphism , the comparison of the distribution of allele ( a , g ) and genotype ( a / a , a / g , g / g ) frequencies in cases and controls by chi - square test was not statistically significant ( respectively , p = 0.49 , p = 0.58 ) . previous studies have shown that cases affected by fhm , carrying mutations of genes encoding ion channels ( cacna1a , atp1a2 , scn1a ) , may have seizures [ 2022 ] . molecular analysis of the cacna1a and atp1a2 genes in patients with migraine without aura and with aura , has shown that these two genes are not involved in common forms of migraine [ 8 , 1113 ] , although similar studies have not examined the scn1a gene . the comorbidity between migraine and epilepsy may allow to select a clinically more homogeneous subgroup of patients in order to carry out genetic studies more targeted than those conducted in patients with common forms of migraine . , carried out on 20 families with migraine and epilepsy , which showed mutations in the atp1a2 gene in 10% of cases , suggesting to perform molecular analysis of this gene in cases where there is comorbidity between migraine and epilepsy and positive family history for migraine or epilepsy . in our study , molecular analysis of five polymorphisms in coding exons of the scn1a gene was performed on a large series consisting of 49 subjects with comorbidity between primary headache and idiopathic or cryptogenic epilepsy and 100 healthy nonmigrainous controls . among the five snps analyzed , only 3199a > g was confirmed to be a polymorphism while the other four snps were not true polymorphisms , because they were not found in the 298 alleles ( cases and controls ) examined and they are not to be further investigated . we did not find a statistical significant difference of 3199 a > g genotypes distribution between cases and controls ; therefore our results confirm that the polymorphism 3199a > g is not associated to pathological phenotypes in headache sufferers and in migraineurs , similar to that found in other studies conducted on patients with epilepsy . from a statistical point of view , the group sample sizes achieve a power lower than 20% to detect , as statistically significant , the observed differences between the two groups for both allele and genotype analyses for the polymorphism 3199a > g. actually , those differences could be considered negligible from a genetic point of view when bearing in mind the epidemiological and clinical characteristics of the disease . furthermore , specifically in reference to genotypes , the observed prevalence would suggest that the a / a genotype is less prevalent among wild type subjects , and this would be inconsistent with expected . for these reasons , in our opinion , the results suggest that the observed differences are due to chance . to our knowledge , this is the first study that has assessed the genotype of polymorphisms in the scn1a gene in cases with comorbidity between headache and epilepsy . the scn1a gene has so far been well studied in large series of patients with severe myoclonic epilepsy [ 2426 ] or in families with idiopathic generalized epilepsies and febrile seizures plus ( gefs+ ) [ 27 , 28 ] and also in some families with febrile seizures . these studies also contain data on the frequency of polymorphism 3199a > g of scn1a gene , and documented genotypic and allelic frequencies comparable between patients and controls . the polymorphism 3199a > g was analyzed specifically in patients with febrile convulsions or with migrainous vertigo excluding a positive correlation . in two recent studies , the analysis of polymorphism 3199a > g of the scn1a gene was conducted in asiatic patients affected by epilepsy [ 32 , 33 ] ; in both studies genotype distributions in drug - responsive and drug - resistant patients did not differ significantly . only in the study of lakhan et al . a statistically significant correlation between the ag genotype of polymorphism 3199 g > a and the presence of epilepsy in cases compared to controls has been found ; however , this finding deserves further study because the statistical significance was observed only in heterozygous subjects , and thus it is more difficult to explain in terms of biological plausibility . overall , the results of the studies conducted so far argue that the polymorphism 3199a > g of the scn1a gene is not associated with pathological phenotypes ; our results confirm that this also applies to headache and migraine sufferers . we found a missense mutation 1811 g > a ( r604h ) in exon 11 in a patient with episodic tension headache and childhood epilepsy with absences ; in the literature this mutation is already known [ 3436 ] . identified the same mutation in two patients with juvenile myoclonic epilepsy and emphasized that this mutation interrupts a consensus site for protein kinase a , which is conserved in the four major sodium channel of the central nervous system . however , this mutation did not cosegregate with the epileptic phenotype in two families with juvenile myoclonic epilepsy and had a minor effect on ionic currents in the study by smith et al . . the possibility that this mutation is not pathogenic has been supported by two subsequent works [ 35 , 36 ] . in our case , the pathogenetic significance of this mutation was excluded by carrying out the genetic analysis of the patient s parents ; in fact the father was asymptomatic and carried the same sequence variation . we also confirm that the missense variant 5782c > g ( r1928 g ) , found in a single healthy nonmigrainous control among the 100 examined , is a rare missense polymorphism , as previously reported [ 27 , 36 ] . our study seems to exclude a role of the scn1a gene in the pathogenesis of migraine and in particular in cases with comorbidity between migraine and epilepsy . the major limitation of the present study is that the genetic analysis has been focused on a part of the gene , since a complete genetic analysis seemed too expensive , given the large number of exons ( equal to 26 ) of the scn1a gene and based on the negative results in previous studies [ 27 , 38 ] . our data support the hypothesis , that the scn1a gene is not implicated in the pathogenesis of common forms of migraine , even when the phenotype is restricted by the presence of comorbidity with epilepsy . the scn1a gene has a causative role in some epilepsy syndromes ( severe myoclonic epilepsy and gefs+ ) and has recently been associated with new and different clinical phenotypes , such as focal and generalized cryptogenic epilepsy and atypical panayiotopoulos syndrome . another important aspect of this study was the use of an innovative method , the hrm , for the molecular analysis of the scn1a gene . the hrm offers significant advantages over traditional methods ( sscp , dgge , dhplc , etc . ) , as it does not require manipulation of genetic material after amplification , it is inexpensive , it has excellent sensitivity and specificity and allows the concomitant analysis of a large number of samples , increasing the reproducibility of analysis and significantly reducing the work time . the sensitivity and specificity of variation detection of hrm these characteristics are of particular relevance , especially in the genetic analysis of multifactorial diseases , such as headache and epilepsy , which requires a large number of subjects and very often involves the analysis of several polymorphisms in the same patients . the hrm could then form the new method of molecular analysis of the scn1a gene , which is currently usually performed with screening techniques ( dhplc ) and direct sequencing . to the best of our knowledge , this is the first study that evaluated the association between polymorphisms of the scn1a gene and comorbidity between primary headache ( especially migraine ) and epilepsy . our results confirm that the polymorphism 3199a > g is not associated with pathological phenotypes in patients with headaches and migraine , while the other four polymorphisms examined were homozygous in the tested populations and should not be further investigated . we exclude the role of the scn1a gene in the pathogenesis of comorbidity between headache ( especially migraine ) and epilepsy . the scn1a gene is a major gene in different epilepsies and epilepsy syndromes , and in this field it has to be further investigated . the hrm could be a new methodology , more rapid and efficacious , for molecular analysis of the scn1a gene . the development of protocols for more efficient and reliable molecular diagnostics would allow a wider appreciation of the role of the scn1a gene and of other genes encoding for ion channels in different epilepsies and epilepsy syndromes .

the purpose of this study was to evaluate the distribution of the polymorphisms of the scn1a gene in a series of children and adolescents with primary headache and idiopathic or cryptogenic epilepsy compared to controls . five non - synonymous exonic polymorphisms ( 1748a > t , 2656 t > c , 3199a > g , 5771 g > a , 5864 t > c ) of the scn1a gene were selected and their genotyping was performed , by high resolution melting ( hrm ) , in 49 cases and 100 controls . we found that among the five polymorphisms , only 3199a > g was a true polymorphism . we did not find a statistically significant difference between distribution of 3199a > g genotypes between cases and controls . we excluded the role of the scn1a gene in the pathogenesis of comorbidity between headache ( especially migraine ) and epilepsy . the scn1a gene is a major gene in different epilepsies and epilepsy syndromes ; the hrm could be the new methodology , more rapid and efficacious , for molecular analysis of the scn1a gene .

Introduction Materials and methods Results Discussion Conclusions

PMC3361345

modified nucleic acids have great potential for applications in oligonucleotide - based drug design . as natural rna and dna molecules are highly sensitive towards nuclease digestion and often possess low thermal stability , great effort has been made to design nucleic acid modifications that stabilize rna or dna while simultaneously maintaining the overall watson - crick base pairing ability . modified nucleic acids are indispensable for future applications comprising diagnostic and clinical approaches like the use of aptamers or the sirna technology . extensive and challenging experiments and investigations have been undertaken to develop nucleotide analogues that maintain the overall a - rna - type conformation and n - type sugar puckering , as such modifications are likely to allow the substitution of rna without large changes in functionality . considerable effort has been made in the synthesis and characterization of 2-o - methyl - rnas , 2-f - rnas , phosphoramidate - rnas , and the locked nucleic acid family . by using locked nucleotide building blocks containing the 2-o,4-c - methylene--d - ribofuranose ( lna ) modification , a significant increase in thermostability can be observed in accordingly substituted rnas . for example , the melting temperature of modified rna helices can be increased by + 2 to + 10c per lna monomer substitution . to understand the stabilizing effects of lna - substituted rnas , numerous structural investigations have been performed during the past years to investigate their conformation in detail . these studies provided insights in the local geometric parameters of mix - mer lna - rna helices and of lna - rna heteroduplexes . the 2-o,4-c - methylene--d - ribofuranose lna - rna mix - mer duplexes maintain mainly the overall a - type nucleic acid conformation . on the other hand , the 2-o,4-c--l - ribofuranose lna modification is used in dna substitution , as this modification preserves the overall b - type nucleic acid geometry of dna . thus , there are two powerful nucleotide modifications with great potential in drug design , the 2-o,4-c - methylene--d - ribofuranose nucleotides ( lna ) for rna substitution and the 2-o,4-c - methylene--l - ribofuranose nucleotides to modify dna . the structure of heteroduplexes , consisting of one fully modified lna strand hybridized to either rna or to dna , revealed the following : the rna conformation is maintained upon hybridizing a 2-o,4-c - methylene--d - ribofuranose lna strand to rna , whereas a mixed n- and s - type sugar puckering is induced by hybridizing a 2-o,4-c - methylene--d - ribofuranose lna to dna . the b - type conformation is maintained by using a 2-o,4-c - methylene--l - ribofuranose lna strand targeted to dna . it is generally accepted that the 2-o,4-c - methylene--d - ribofuranose locks the lna in the c3-endo conformation . this approach is used to direct the geometry of the phosphate backbone in a manner to orient the duplex towards a more efficient base stacking . even though the 2-o,4-c - methylene--d - ribofuranose lna - rna mix - mer duplexes maintain the overall a - type nucleic acid conformation , molecular dynamics simulations and a crystal structure of all lna duplexes , consisting exclusively of 2-o,4-c - methylene--d - ribofuranose building blocks , yielded insights into a novel nucleic acid geometry . an all lna duplex shows alterations in the local and overall helical parameters as compared to natural rna and can rather be compared to other modified nucleic acids , like glycol nucleic acids ( gnas ) , peptide nucleic acids ( pnas ) , or homo - dna . an lna duplex appears as a right - handed , antiparallel helix that maintains the canonical watson - crick base pairing and the 2-exo conformation for all nucleotides . nevertheless , the lna duplex shows a considerable decrease in the helical twist , roll and propeller twist , which facilitates a widening of the major groove and a decrease of the minor groove dimensions . these alterations induce a large hollow cave in the middle of the duplex that is obvious in a projection perpendicular to the helical axis . due to an enlarged helical rise and the unwinding of the helix , which results from the decrease in the twist angle parameters , the lna duplex possesses an increased helical pitch . the unique nucleic acid geometry of all lna helices apparently induces a more efficient and stable base stacking , which contributes to the higher thermostability of lnas and lna - modified nucleic acids . interestingly , the structure of an rna / lna heteroduplex is a geometric intermediate between the rna and the all lna conformation . the 7mer lna helix was derived from the e. coli trna isoacceptor with the data base compilation of trna sequences and sequences of trna genes i d rs 1661 and represents the sequence of the trna aminoacyl stem microhelix that has been crystallized previously possessing the sequence 5-(g - g - u - g - a - g - g-)-3 and 5-(c - c - u - c - a - c - c-)-3 . the base sequence of the rna was maintained for further comparative studies , except for the u to t and the c to mc exchange used in standard lna synthesis . the chemically synthesized single strands with the sequences 5-(g - g - t - g - a - g - g)-3 and 5-(mc - mc - t - mc - a - mc - mc)-3 were purchased from iba ( gttingen , germany ) with hplc purification grade . crystals were grown within 3 - 4 days using 40 mm sodium cacodylate , ph 5.5 , 20 mm cobalt hexamine , 80 mm sodium chloride , 20 mm magnesium chloride , and 10% ( v / v ) mpd with equilibration against 1 ml 3341% ( v / v ) mpd at 21c using the hanging drop vapour diffusion technique . data collection of the lna crystals was performed at the elettra synchrotron ( trieste , italy ) beam line xrd-1 at a wavelength of 1.0 and a temperature of 100 k. the crystal diffracted up to 1.9 resolution . the corresponding trna - microhelix was measured at the desy synchrotron ( hamburg , germany ) at a wavelength of 0.8123 , 100 k temperature , and diffracted up to 1.2 . molecular replacement calculations were performed using the program phaser within the ccp4i program suite . the lna structure was solved by using a model built from the previously solved trna microhelix structure but exchanging the riboses by 2-o,4-c - methylene--d - ribofuranose residues . standard lna nucleotides were used for model building , which comprises the standard u to t and c to mc substitutions in lna as compared to rna . refinement calculations were done applying the program refmac , and electron density maps were calculated using fft , as implemented in the ccp4i package . structure representations and graphical analysis of helices were performed with the programs coot and pymol . we analysed the crystal structure of a locked nucleic acid duplex , which contains exclusively 2-o,4-c - methylene--d - ribofuranose nucleotides ( figure 1 ) , in comparison to the structures of the naturally occurring rna as well as to other backbone - modified nucleic acids like glycol nucleic acids ( gnas ) or peptide nucleic acids ( pnas ) . the lna helix structure reveals a nucleic acid duplex geometry that significantly differs from the canonical a - type rna structure ( figure 2 , tables 2 and 3 ) . the structure of the lna duplex appears as a stretched helical ladder with altered local and overall geometric parameters . the observed geometry can be rather compared to that of glycol nucleic acids ( gnas ) , peptide nucleic acids ( pnas ) [ 26 , 27 ] , or homo - dnas . we detected a notable decrease in several local and overall helical parameters in the lna helix , like the twist , roll and propeller twist , as compared to a corresponding rna molecule ( table 2 ) . this results in a widening of the major groove , a decrease in helical winding and an increased helical pitch . the major groove dimensions in the lna duplex showed values of around 24 - 25 in diameter , as compared to 16 observed for the canonical a - rna duplex . concomitantly , the minor groove of lna duplexes is narrower ( about 15 ) than that of standard rna helices ( 19 ) . on the other hand , the slide and rise values are slightly increased in the lna helices . moreover , the shift of the base pairs in the lna duplex results in an empty tunnel running through the center of the helix . in the lna helix , the low twist angle of 26 and the large pitch of 14 base pairs per turn lead to an unwinding of the duplex , as compared to rna , which possesses a twist of 32 and a pitch of 11 base pairs per turn . the helical rise of lna falls into a range of 2.83.0 , whereas the helical rise in rna is 2.6 . due to the increased rise and the unwinding of the helix , the lna possessed an enlarged helical pitch of 39 , as compared to 29 in rna helices . the backbone torsion angles resembled the sc , ap , sc , sc ap , sc , and ap conformation for the , , , , , , and angles with the sugar puckering being in the 2-exo conformation . the phosphate - phosphate distances are in the region of 5.6 as compared to 6.0 for rna . it is conceivable that the altered helical parameters in lna duplexes provide an enhancement in nucleotide stacking , leading towards stronger - interactions of the base pairs . it is well accepted that the extensive hydration of the rna minor groove plays an important role in the structure / function relationship . as the specific hydration pattern of rna is governed by the 2-hydroxyl group , it has been questioned whether the 2-o,4-c - methylene--d - ribofuranose in lna allows a comparable hydration as described for rnas . therefore , we focussed our investigations on analyzing the arrangement of the solvent molecules surrounding the lna duplex . we observed that the distribution of water molecules in the lna minor groove follows the general pattern known for rna hydration , as the bridged 2 oxygen atoms in the 2-o,4-c - methylene--d - ribofuranose moieties serve as hydrogen bond acceptors similar to the 2-hydroxyl residues in rna . interestingly , the structure of an rna / lna hybrid helix represents a geometric intermediate between rna and all lna helices . in figure 4 , we present the structure of idealized rna as compared to the rna / lna hybrid ( pdb i d : 1h0q ) and to the all lna duplex ( pdb i d : 2x2q ) as calladine - drew plot . to better visualize the overall geometry in this figure , the helices were extended to 22 base pairs for rna , to 25 base pairs for rna / lna , and to 28 base pairs for lna showing two full helical turns each . for an overall comparison of a - rna and b - dna helices to the conformation of different backbone - modified nucleic acid types , like gna and pna ( figure 5 ) , we displayed the selected nucleic acid helices with a total length of 46 base pairs ( figure 6 ) . we illustrate the natural dna and rna and the synthetic gna ( pdb i d : 2jja ) and pna ( pdb i d : 1pup ) as compared to the lna ( pdb i d : 2x2q ) duplex structure . the standard a- and b - type nucleic acid conformations are paraphrased by the rna and dna helices . the gna shows the structure of a helical ribbon with only one large minor groove and completely lacks the major groove , which is instead a convex surface . the pna resembles the helix with a wide and deep major groove concomitant with a narrow and shallow minor groove [ 26 , 27 ] . the weakly twisted right - handed homo - dna structure has been described to explain the inability of allo- , altro- , and glucanosyl - nucleotides to form stable base pairing systems ( picture not shown ) . in the middle of figure 6 we present the extended structure of the lna duplex , which represents the unusual geometry , which can rather be brought into vicinity of gna , pna , and homo - dna than to the natural nucleic acid duplexes dna and rna . the lna helix , however , possesses a pitch of 39 with 14 base pairs per helical turn and an average rise of 2.8 . dna and rna show an average pitch of 30 and 34 , respectively , as compared to mean values of 60 , 58 , and 53 found for gna , pna , and homo - dna . ( figure 6 , table 3 , no data shown for homo - dna ) . conclusively , regarding the geometry of the lna duplex in total , this helix resembles a more natural nucleic acid , when compared to the other backbone - modified duplexes . as has been reviewed , an increase of the melting temperature between + 2 to + 10c can be observed per lna building block added in strands hybridized to rna . the short 7 bp lna duplex , derived from the trna microhelix for this study , exhibits a melting temperature of above 90c , whereas the corresponding rna has a tm value of 45.0c . we have previously investigated the melting temperature of another lna 7mer helix in comparison to its natural rna counterpart . similarly , the lna duplex possesses a tm value of 84.3c , whereas its corresponding rna helix melts at 22.4c . the drastic shift in thermostability in both lnas as compared to the rnas corresponds to an average of 4.5c per nucleotide building block , which is consistent with the reviewed observations . thus , the thermostability data and the structure properties of lnas provide new perspectives for future nucleic acid drug applications , which is an encouraging outlook . considering that the increase in tm values by substituting natural nucleic acids with single or multiple nucleotides by lna residues seems to be a summative property nearly any natural nucleic acid can be modified by introducing single and multiple lna building blocks or even complete lna duplexes , thereby stepwise increasing the thermostability depending on the number of introduced lna residues . depending on the particular requirements , any nucleic acid can thus be stabilized at will with little or no loss of function . in this respect , lna substitution may serve as a reliable method to stabilize nucleic acids , in particular aptamers , for clinical applications . an upcoming challenge is to stabilize aptamer stem regions by introducing lna portions without affecting the loop regions that are usually essential for target binding and specificity . several reports in the literature highlight a forthcoming application of lna - substituted aptamers with retained or even improved ligand - binding capacity . an exciting example is the use of lna modifications within hammerhead ribozymes that improve the overall cleaving capacity . in addition , lna modifications have successfully been introduced into antisense oligonucleotides and dnazymes that were targeted to functionally selected binding sites and inhibited hiv-1 expression . a third example describes a g - quadruplex thrombin aptamer , which retained the biological activity to a varying extent depending on the nucleotide positions that were lna modified . these selected reports are a snapshot of numerous studies demonstrating the great potential of lna substitutions in functional nucleic acids and possible therapeutic applications . the crystal structure of the all locked nucleic acid helix contributes to the understanding of the structure / function relationship and the high thermostability of these molecules . in summary lnas possess an encouraging potential for the development of new stabilized nucleic acids and will promote future applications in diagnostics , drug discovery , and clinical therapy .

locked nucleic acids ( lnas ) belong to the backbone - modified nucleic acid family . the 2-o,4-c - methylene--d - ribofuranose nucleotides are used for single or multiple substitutions in rna molecules and thereby introduce enhanced bio- and thermostability . this renders lnas powerful tools for diagnostic and therapeutic applications . rna molecules maintain the overall canonical a - type conformation upon substitution of single or multiple residues / nucleotides by lna monomers . the structures of all lna homoduplexes , however , exhibit significant differences in their overall geometry , in particular a decreased twist , roll and propeller twist . this results in a widening of the major groove , a decrease in helical winding , and an enlarged helical pitch . therefore , the lna duplex structure can no longer be described as a canonical a - type rna geometry but can rather be brought into proximity to other backbone - modified nucleic acids , like glycol nucleic acids or peptide nucleic acids . lna - modified nucleic acids provide thus structural and functional features that may be successfully exploited for future application in biotechnology and drug discovery .

1. Introduction 2. Material and Methods 3. Results and Discussion

PMC3426189

the ability to reproducibly and accurately quantify proteins or proteomes is important for life science research , and the recent development of targeted proteomics strategies , i.e. , selected reaction monitoring ( srm ) , greatly increases the quantitative reproducibility and accuracy compared to conventional data - dependent mass spectrometry analysis . recent srm workflows have displayed a large linear dynamic range and high quantification reproducibility using stable isotope standards ( sis ) . in srm , proteotypic peptides are quantified in a triple quadrupole mass spectrometer by measuring specific peptide ions and some of their most specific and most frequently appearing fragments . the combination of a peptide ion and fragment ion mass - to - charge is called a transition . gathering of the a priori information of what peptides and fragments to target , called srm assays , has been a bottleneck in srm analysis , but the presence of large peptide identification repositories such as peptideatlas , recent advances in computational prediction algorithms , the use of crude synthetic peptides , and the ongoing construction of srm atlases are rapidly decreasing srm assay development time . furthermore , throughput of the method is increasing with the introduction of scheduled srm and isrm , presenting the possibility to use srm for screening complete pathways and even complete microbial proteomes . however , examining this new larger scope reveals two new bottlenecks : stable isotope labeling , which effectively halves ms throughput , increases sample preparation complexity , and is normally associated with long synthesis times and high cost , and manual data analysis , which limits routine high throughput and introduces bias . previous studies have reported that label - free quantitative srm may generate data of sufficient quality for analysis of biological samples . however , increasing the number of target proteins also requires automated data analysis , and some objective measure of quality for each detection to control false discoveries . of previously published software , mprophet and the ddb workflow perform detection and quantification of peptides in srm data in an automated fashion while providing custom scores for quality control but in return have the drawbacks of relying on decoy measurements and requiring a large assay database respectively . most current srm software ( skyline , pinpoint , mrmer ) focus on assisting in construction of srm assays , presenting the data , and assisting manual quantification . skyline does perform detection and quantification , but without any clear measure of quality , and in addition requires a spectral library . the audit software assists manual data analysis by highlighting peptides with large variation in sis - corrected quantity or deviating fragment ratios between endogenous and sis peptide . in this report we present a novel algorithm that does not rely on decoy data or spectral libraries but focuses on label - free srm , estimates the quality of each reported detection , and supports interference correction during quantification , to circumvent the drawbacks of using sis - labeling and manual data analysis . we demonstrate that the algorithm , called anubis , performs srm data analysis on par with a human expert , elaborate on the reproducibility and accuracy achievable with the anubis label - free workflow , and apply it to study the effect of human plasma on a set of targeted streptococcus pyogenes proteins . anubis is vendor independent , accepts mzml data files and traml or csv transition lists , is operating system independent by being implemented in scala 2.8.0 , and is run in a java virtual machine . source code , compiled binaries , and usage instructions are available at http://quantitativeproteomics.org/anubis/ under an open source license . the streptococci used were grown from a single colony of s. pyogenes strain sf370 . this culture was sampled into 2 sets of 10 replicates , where one set was grown in pure todd - hewitt broth ( th ) and one in th supplemented with 10% citrate treated human plasma ( skne university hospital , sweden ) . cells were grown to exponential phase ( od620 = 0.5 ) and were harvested , suspended , and lysed using standard procedures . protein concentrations were estimated with pierce coomassie protein assay kit ( 10 l sample to 240 l reagent in duplicates , 96-well plate , a595 victor ) , showing a reduced yield in one th sample ( supplementary table 5 ) . the samples were prepared for srm by taking 50 l of each harvested culture and adding 2.5 l of 1 pmol/l adh1_yeast ( adh ) , after which 0.6 l of 0.5 m tcep was added and samples were left to incubate in 37 c for 1 h. after adding 1.2 l of 500 mm iodoacetamide , samples were left in the dark for 45 min , 2 l of 0.5 g / l trypsin was added , and samples were incubated in 37 c overnight . samples were desalted using c18 columns ( the nest group , southborough ) , dried in vacuo , and resuspended in 50 l of 2% acetonitrile ( acn ) , 0.2% formic acid ( fa ) , by sonication for 5 min , followed by centrifugation at 3,100 rcf for 30 s before transferring the supernatant to hplc vials . srm assays were created using synthetic peptides ( spiketides , jpt peptide technologies gmbh ) . in total 163 peptides originating from 41 proteins the dried peptides ( approximately 50 nmol ) were dissolved by addition of 180 l of 20% acn , 1% fa to each peptide well , sonication for 5 min , and shaking for 1 h. five microliters was sampled from each well and pooled . the pool was dried out and redissolved in 450 l of 2% acn , 0.1% fa , and 100 l was transferred to an hplc vial . for each synthetic peptide 1015 transitions were generated in skyline , and these were measured with unscheduled srm . the resulting data were used to manually reduce the number of transitions per precursor to a maximum of 5 , choosing the transitions with highest intensity and with not more than one transition with a product m / z smaller than the m / z of the precursor . the retention times of the synthetic peptides were used to generate a final scheduled , 5 min window , srm method . all ms analysis was carried out on a tsq quantum vantage ( thermo - fisher scientific , waltham ma ) triple quadrupole instrument interfaced with an eksigent nanolc 1dplus lc system ( eksigent technologies , dublin ca ) . the mobile phase consisted of solvent a , 0.1% aqueous formic acid and solvent b , acetonitrile with 0.1% formic acid . peptides were separated on a 10 m tip , 75 m 12 cm capillary column ( picotip emitter ) packed with reprosil - pur c18-aq resin ( 3 m , dr . the system was washed and equilibrated in a separate water injection between each sample injection . sample injections were 1 l at a constant flow of 300 nl / min , with a gradient of 97% solvent a at 05 min , 85% a at 8 min , 65% a at 42 min , 10% a at 4550 min . tsq cycle time was 1 s and q1 and q3 peak widths of 0.7 m / z . instrument raw files were converted to mzml using msconvert from proteowizard . the median protein group cv was calculated as the median of the cvs of all the peptides measured for proteins in the group . for detecting significant differences in center between or two biological conditions , we have used a combination of student s t test and wilcoxon s rank sum test , both two - sided . q - q plots of some arbitrary peptides show that for peptides with reasonably low cv , replicate measurements are roughly normally distributed , although one high cv peptide demonstrated typical non - normality ( supplementary figure 24a , b ) . because of this we consider differences significant only if both the parametric and nonparametric test show significant difference at p 0.05 . with the high total numbers of mass spectrometry analyses in this study , we have excluded replicates because of column failure , failed protein extraction , large synthetic peptide carry - over , and in one case unexplainably low total signal . all exclusions are reported in supplementary table 2a , b and supplementary figure 7 . normalization of label - free s. pyogenes data was done using a house keeping protein index ri defined as the average peptide quantity of rs10_stra1 , rl22_strp1 , rl1_strp1 and rs17_stra1 proteins in replicate i. peptide quantities in replicate i was divided by ri and multiplied by the average ri across the replicates . the original data in this work has been deposited at the swestore repository : http://webdav.swegrid.se/snic/bils/lu_proteomics/pub/anubis_data.zip peptide quantification using srm is typically performed by measuring multiple transitions to ensure that the signal is derived from the target peptide . the chromatographic traces of the measured transitions should all display a peak when the peptide elutes , and furthermore the ratios of the peak intensities should be identical to previously measured peaks of the same peptide , unless the transitions are contaminated by other compounds . the anubis algorithm was tailor - made for these properties and works by comparing chromatograms from complex biological samples to user - provided reference ratios to locate the target peptide . conventionally , chromatogram analysis is performed by searching for peak - shaped sections in each fragment ( e.g. , by local maxima or by a moving reference shape ) , clustering of the peak shapes , and then selecting the best cluster using some heuristic , often based on intensity , rank between fragments , or retention time . in anubis , we have used a novel approach in which possible elution points are searched for in the pairwise fragment ratios rij of the chromatogram , by comparing to target pairwise fragment ratios , tij . for a given instrument , collision energy and peptide , the frequency of each fragment is constant , and we can therefore simply search for retention times where a rij agrees with the respective tij ( figure 1a d ) . ( d ) in the data , every time point during which any pairwise fragment ratio is close enough to its target is marked as a possible peak of peptide elution . ( e ) using wavelet analysis , p - values are estimated for each possible peak , and the most specific peak is chosen . ( f ) again the target pairwise fragment ratios are used to remove any substantial interference , which gives the final peak . this ( g ) we validated anubis with a large srm study of 8 laboratories , 10 peptides , and a 9-point dilution series . using sis references , anubis achieves equal accuracy as the previously published manual analysis . ( h ) label - free anubis analysis gives still accurate , but slightly reduced , performance . to allow assessment of the quality of reported peptide quantities , a local p - value is estimated for each detected peak , allowing the filtering of a data set at any confidence level . we calculate p - values by deconstruction and reassembly of the chromatogram using wavelet analysis , in a way that preserves the general frequency content of each fragment but removes any correlation between fragments , thus generating random chromatograms with properties similar to the original chromatograms . this is performed 1,000 times to create a null distribution , and the p - value is taken as the fraction of the null distribution where there is a point of equal or better agreement between rij and tij compared to that of the detected peak , thus indicating a false discovery . in addition to providing the user with a sense of the peak detection quality , the p - value is also used to pick the most specific ( lowest p - value ) peak if there are multiple possible peaks in a chromatogram . quantification is done by summing the fragment areas , while excluding interference in fragments that are not part of any agreeing rij ( figure 1e , f ) . the size of the null distribution was chosen as a trade - off between analysis time and p - value precision ( supplementary results ) . reference ratios are preferably derived from a chromatogram with a clear peak of the target peptide , using the reference creator program that is supplied with anubis . for peptides from naturally high abundance proteins , reference ratios can often be measured directly in the biological sample , when unambiguous peaks exist . for peptides where the correct peak is not readily distinguishable in the biological sample , , hundreds of crude synthetic peptides can be pooled and analyzed for measuring reference chromatograms in a minimum of instrument time . as an alternative to synthetic peptides , in vitro synthesized proteins only the pairwise fragment ratios are needed from the reference chromatogram , another approach that we have not evaluated would be to calculate the ratios directly from spectral libraries , but for accurate results the library would need to be acquired under similar instrument settings as in the final srm method . note that because anubis uses fragment ratios , a theoretical minimum of 2 transitions per peptide is needed , but we generally find that 3 transitions are necessary for reliable detection , and at least 4 transitions are needed for maximal accuracy in the quantification ( supplementary results ) . the design of the anubis algorithm has a number of advantages . compared to creating peak candidates from a cluster of local intensity maxima , looking directly at the pairwise ratios allows evaluation of each fragment at each individual time point , giving a much more refined representation of the peak . this makes exclusion of ill - behaving fragments possible , whereas the dot product between the relative ratios and a spectral library or other aggregate measures are incapable of this . as the signal - to - noise decreases the fragment ratios remain constant ( supplementary figure 5 ) , meaning that peaks will be detected equally well , but their assigned p - value will be higher since similar peaks will occur more frequently in the null distribution . finally , we have chosen not to utilize retention time in our analysis , since retention times typically fluctuate and column degradation can give systematical shifts in large sample batches ( supplementary figure 6 ) , which means that deviations in raw retention time contain little information for distinguishing target peaks within the small time windows typically used in scheduled srm . retention time has indeed been shown to be the least discriminant dimension of information in srm . efficient usage of retention time for peak discrimination requires either some efficient means of retention time normalization or inter - replicate analysis , which we prefer to leave outside the core algorithm since this is heavily experiment setup dependent . in addition to the aim of extracting the most information possible out of srm data , anubis was specifically designed for being easy to use , for software pipeline integration , and to support high throughput . we support the standard file formats mzml and traml , as well as transition lists exported from skyline . the software is platform independent , and analysis is easily automated using the command line interface . although the need for reference ratios could imply a potential lowering of throughput if synthetic peptides are used as references , these analyses only have to be done once per peptide and are typically performed during assay development regardless . we validated anubis performance in a label - free quantitative workflow against a large previously published multisite data set . this data set consists of 10 peptides diluted into human plasma over 3 orders of magnitude , and the resulting sample set was analyzed at eight different laboratories followed by expert manual data analysis . we reanalyzed this data with anubis and compared coefficients of variation ( cv ) and coefficients of determination ( r ) with values reported in the original publication ( supplementary table 1 ) . since the study was made using sis labeling , we quantified both endogenous and sis peptides with anubis and calculated statistics for both endogenous quantities and endogenous divided by sis quantities . anubis sis reference statistics match the manually analyzed results , showing the validity of the algorithm and its ability to perform unsupervised analysis of complex srm data ( figure 1 g ) . statistics for anubis quantities with or without sis labels shows that labeling is beneficial for accurate srm quantification as expected , but label - free quantification is still reliable , demonstrating that the label - free anubis workflow allows accurate quantification ( figure 1h ) . we further confirmed the validity of the anubis workflow by performing spike - in experiments with a dilution series of 42 synthetic peptides in a cell line lysate using skyline and automated anubis analysis in parallel ( supplementary results ) . although performing well on the spike - in data sets , the utility of the label - free quantification workflow needed to be confirmed on real biological experimental data , without extensive assay optimization . we thus conducted a series of label - free experiments on s. pyogenes , an important microbial pathogen often responsible for pharyngitis but occasionally causing severe conditions such as septic shock . s. pyogenes is responsible for more than 500,000 deaths worldwide , making it one of the most important human pathogens . we cultured 9 biological replicates of s. pyogenes ( supplementary table 2a ) , with replicates grown , harvested , and prepared for srm in parallel to minimize the biological and experimental variation . from a previously published s. pyogenes instance of peptideatlas , we selected 10 s. pyogenes ribosomal ( rib ) proteins , 14 fatty acid synthesis ( fas ) pathway proteins , and 29 virulence associated or presumed virulence associated proteins ( virulome ) representing a complete coverage of the intracellular dynamic protein abundance range . for these proteins synthetic peptides were made for one to three previously identified proteotypic peptides , and reference chromatograms were established by analyzing pools of the synthetic peptides by srm , giving validated assays for 161 peptides ( supplementary table 3 ) . to assess the overall relationship between the technical and total variability ( technical plus biological variability ) in label - free srm using anubis , we processed and quantified the 9 biological replicates with the anubis workflow , giving total variability median cvs of 18% , 19% , and 38% for the respective protein groups ( rib , fas , and virulome ) ( figure 2a ) . to estimate the influence of sample preparation steps , srm measurement , and data analysis , we made up to 10 repeated measurements on singular biological replicates , giving six technical replicate sets of totally 56 successful replicates ( supplementary table 2b , supplementary figure 7 ) . the median cvs for these six sets were 411% , 715% , and 1526% for the protein groups . compared with the biological replicate cvs ( figure 2a ) , the technical cvs are consistently smaller by about half , meaning that only 1/4 of total experimental variability originates from the mass spectrometer analysis and the anubis label - free workflow . we therefore estimate the technical srm variability to be considerably lower than the total variability in bacterial samples with minimized biological variability and that the accuracy of quantification of the label - free srm workflow is sufficient for analysis of complex bacterial samples . proteins are divided into ribosomal ( rib ) , fatty - acid synthesis ( fas ) , and virulence - associated proteins ( virulome ) , which are respectively high- , medium- , and low - abundant . ( a ) median cvs across the peptides in 3 protein groups , for 6 10 technical replicates and 9 biological replicates . ( b ) median cv ranges for the 6 10 technical replicates compared to median cv ranges 2 sets separately prepared replicates ( a2.1apr + a2.2apr and a2.1jun + a2.2jun ) . ( c ) median cv ranges for the 6 10 technical replicates compared to median cv ranges of 2 sets replicates analyzed at different times ( a2.1apr + a2.1jun and a2.2apr + a2.2jun ) . ( d ) median cvs with interquartile ranges for the technical replicate sets compared to all second batch replicates ( a2.1apr , a2.2apr , a2.1jun and a2.2jun ) , with and without normalization . ( e ) illustration of normalization by ribosomal housekeeping proteins ( rs10_stra1 , rl22_strp1 , rl1_strp1 , and rs17_stra1 ) in one biological replicate . each row represents a peptide , and each column a replicate grouped into 5 sets ( supplementary table 2b , supplementary data 1a - b ) . color denotes the quantity of the peptide in that replicate compared to its average across all replicates . from comparing a2.1apr + a2.2apr to a2.1jun + a2.2jun it is clear that the time of analysis greatly affects the measured quantity . after normalization these differences complementary to single preparation back - to - back technical replicate sets , combinations of sets allowed for investigation of the experimental variability caused by sample processing and instrument condition at the time of injection . combining replicates from double sample preparations gave a moderate increase in total variation ( figure 2b ) , while combining replicates from separate times of injection resulted in more substantial variation ( figure 2c ) . however , both of these systematic replicate - wide increases in variability can be negated by proper normalization , which was done by housekeeping proteins since total ion current normalization is typically not possible in label - free srm . normalization by four stable ribosomal proteins gave cvs almost level with the ideal technical replicate sets and allowed combination of data from different times of analysis and multiple sample preparations ( figure 2d , e ) . we also calculated the average amount of successful detections in detectable peptides ( with more than one successful detection ) for each of the six technical replicate sets and the nine biological replicates ( in total 65 injections in seven sets ) . the reproducibility of the sets was high , with rib , fas , and virulome proteins having an average success rate of 97100% , 95100% , and 7584% ( figure 2e ) . closer inspections of virulome peaks revealed that the limited detection of these peptides can largely be attributed to very low signal - to - noise ratios for these peptides , resulting in loss of detection in some injections ( examples are shown in supplementary figures 1322 ) . we believe that the above demonstrates that automated analysis of label - free srm data using anubis possesses the required properties for targeted quantitative proteomics . the technical variability ( 426% ) is considerably smaller than the biological and other experimental variability ( 1838% ) . with high reproducibility of detection ( 75100% ) it presents a way of increasing throughput when absolute quantification is not required . to finally demonstrate the feasibility of anubis , we compared the measurements of our 9 biological replicates grown in standard medium to 9 biological replicates grown in the presence of 10% human plasma ( supplementary table 2a ) , as adaption to human plasma is an important ability for s. pyogenes virulence . growing s. pyogenes with plasma changes the proteome homeostasis of the bacterium but also represents a vastly different sample as there are a substantial amount of plasma proteins present . nevertheless , the increased sample complexity barely influenced the technical variability ( data not shown ) . looking at agreeing wilcoxon rank sum tests and students t tests , we find that the entire fas network is significantly down - regulated by about 40% in the plasma condition ( figure 3a ) , which is supported and explained by previous research . meanwhile , in the highly abundant and therefore easily measurable ribosomes , only one protein is significantly regulated . in the virulome proteins , no group - wide trend is seen , but multiple proteins show significant regulation in all measured peptides . for example , measured up - regulation of c5a peptidase ( figure 3b ) upon plasma exposure agrees with previous results , as well as the suspected down - regulation of streptopain ( figure 3c ) . polyphosphoribitol subunit 1 and 2 ( dltc and dlta , figure 3d ) is expected as they share the same promoter region . these biological findings have been discussed previously by others , but the agreement of our results with previous studies further validates the performance of the label - free setup and automated analysis . in summary , application of label - free srm on s. pyogenes to study protein abundance differences upon growth with human plasma supplement . ( a ) each row represents one peptide , with peptides grouped into proteins and separated by a white row . 0% plasma and 10% plasma columns display measured quantities in the 2 9 biological replicates grown with and without 10% plasma ( supplementary data 2 ) . wilcoxon and t test columns show the p - values of wilcoxon rank sum tests and t tests between conditions , with light green indicating significance 0.05 . the fold change column shows the means ratio between samples , with blue being down - regulated in plasma and red up - regulated . while the high - abundant ribosomes show almost no regulation , indicating that they are not affected by plasma , almost the entire fas ii pathway is significantly down - regulated in plasma by about 40% . ( b ) the virulome protein c5a peptidase shows consistent significant up - regulation in all peptides . ( c ) streptopain is reliably detected in 0% plasma samples but not at all in 10% plasma samples , indicating down - regulation beyond our limit of detection . ( d ) d - alanine polyphosphoribitol ligase subunit 1 and 2 both show significant down - regulation in 10% plasma . the maturing techniques of targeted mass spectrometry have been demonstrated and utilized in numerous reports , typically using isotope labeling and with limited numbers of proteins , samples , and replicates . the major advantages of the anubis workflow are automated analysis , with performance equal to a human expert , and the omission of costly labeling . we still retain a median technical variability of 520% for the label - free workflow in a large scale experiment , and application on s. pyogenes yields significant biological results in agreement with previous data . the measured trend of inverse correlation between abundance and technical variability agrees with previous work , and manual inspection of low abundance peaks indicated that the vast majority of these were correctly assigned and quantified by anubis . we believe that this higher variability of low abundance peptides is naturally close to the limit of quantification , arising from fluctuations in chromatography and ionization , as well as from stochastic ion detection . in a recent smaller scale study by zhang et al . , technical cvs of 10% for sis - labeled srm and 2030% for label - free srm were found , which is slightly above our values . they argue , however , that when performing measurements on clinical samples , the inherent biological variation in clinical material is large enough that a technical variability of even 20% will barely affect the total variability of the experiment . label - free srm has slightly different and complementary characteristics compared to other lc the classical data - dependent shotgun strategy has with recent advances in protocols , chromatography , and ms instrumentation been able to reach both high proteome coverage and high sensitivity . however , it often lacks the ability to reproducibly quantify a given analyte in multiple samples , due to both stochastic ms / ms sampling and difficulties in precursor ms data analysis . several approaches using data - independent ms / ms acquisition have also been proposed , as discussed elsewhere . for the recent variant swath - ms , a data analysis workflow has been proposed where fragment ion chromatograms are extracted and analyzed on the basis of previously acquired ms / ms data , in a manner similar to srm data analysis . this offers an alluring compromise between the srm and shotgun strategies , mimicking the reproducibility and sensitivity of srm but at a lc ms / ms - like throughput . in this initial work , the quantitative reproducibility of swath - ms was indeed comparable to that of srm . it remains to be seen whether this high performance can be repeated on a standard basis , but we can note that swath - ms data potentially could be analyzed using anubis . whether to use an srm strategy with or without labels for any given experiment will always be a trade - off between required accuracy , cost , and instrument time . if measurements have to be made at different time points , or if heterogeneity between biological samples is so large that normalization is troublesome , a strategy incorporating labels might be the only way to control instrument variation . if on the other hand large amounts of analytes need to be measured and the experimenter has much control over how and when , a label - free approach will give similar results faster and at a reduced cost . we believe our proposed workflow enables larger scale srm experiments , with higher throughput , reduced cost , more consistent data analysis , and controlled error rates . all of this boils down to the possibility to target more proteins or using more replicates for additional statistical power , while relieving some highly qualified srm expert of hours of daunting peptide integration . once a small set of highly interesting proteins is found , the synthesis of sis is of course possible and will result in further decreased experimental variability .

selected reaction monitoring ( srm ) is a mass spectrometry method with documented ability to quantify proteins accurately and reproducibly using labeled reference peptides . however , the use of labeled reference peptides becomes impractical if large numbers of peptides are targeted and when high flexibility is desired when selecting peptides . we have developed a label - free quantitative srm workflow that relies on a new automated algorithm , anubis , for accurate peak detection . anubis efficiently removes interfering signals from contaminating peptides to estimate the true signal of the targeted peptides . we evaluated the algorithm on a published multisite data set and achieved results in line with manual data analysis . in complex peptide mixtures from whole proteome digests of streptococcus pyogenes we achieved a technical variability across the entire proteome abundance range of 6.519.2% , which was considerably below the total variation across biological samples . our results show that the label - free srm workflow with automated data analysis is feasible for large - scale biological studies , opening up new possibilities for quantitative proteomics and systems biology .

Introduction Experimental Section Results and Discussion Conclusions

PMC3092601

the increased incidence of obesity in both developed and developing nations is a major public health problem . altered energy balance resulting in obesity is believed to be causal in the development of the metabolic syndrome and type-2 diabetes mellitus , conditions whose incidence is also rising at alarming rates [ 2 , 3 ] . is currently in excess of $ 200 billion annually , and it is expected to rise . diet and lifestyle modifications are often unsuccessful in reducing obesity [ 46 ] , and presently available pharmacologic treatments are problematic due to serious adverse effects . evidence that interleukin-15 ( il-15 ) can inhibit adipose tissue deposition in both laboratory animals and human subjects will be reviewed . the specific receptor for il-15 , il-15 receptor - alpha ( il-15r ) , appears to regulate il-15 secretion , stability , and activity [ 8 , 9 ] . several single - nucleotide polymorphisms ( snps ) in human il15 and il15ra have been described which correlate with adiposity and markers of the metabolic syndrome [ 1012 ] . these findings suggest a model in which genetic variation in il15ra could modulate il-15 activity and bioavailability , which in turn regulate adiposity . moreover , because il-15 mrna is highly expressed in skeletal muscle tissue , some authors have suggested that il-15 may function as a muscle - derived endocrine factor , or when human genetic , cell culture , and laboratory animal studies are taken together , it is clear that the il-15/il-15r axis regulates adiposity . modulation of this axis should be explored a novel target for control of obesity in the human population . il-15 is a 14 kda cytokine that was originally isolated on the basis of its ability to support natural killer ( nk ) t - lymphocyte proliferation . il-15 was subsequently found to be expressed by monocytes , macrophages , and other cell types involved in immunity and to regulate a variety of processes comprising both innate and adaptive immunity reviewed in . il-15 exhibits both pro- and anti - inflammatory actions in a variety of tissues and has both positive anticancer effects via stimulation of nk cells , and deleterious effects such as involvement in inflammatory bowel disease ( reviewed in [ 18 , 19 ] ) . inasmuch as the present paper is focused on the relationship of il-15 to obesity , the reader is referred to comprehensive reviews of the diverse functions of il-15 provided by fehniger and caligiuri and budagian et al . . il-15 is structurally part of the 4-helix bundle protein family , whose members exhibit structural , but not necessarily sequence , homology . this family of proteins includes many cytokines which regulate the immune system and also includes factors with actions outside of the immune system , such as il-6 , leptin , growth hormone , and erythropoietin . il-15 is expressed at the mrna level in a variety of nonlymphoid tissues , with particularly high expression in skeletal muscle and placenta . il-15 is also expressed abundantly in cardiac muscle , lung , liver , kidney , brain , and pancreas . in the placenta , il-15 regulates a specific subset of nk cells involved in endometrial decidualization . in nonlymphoid tissues , conflicting reports [ 14 , 24 ] exist regarding il-15 mrna expression in cultured adipocytes ( reviewed below ) , and a systematic study of il-15 expression in adipose tissue in vivo has not been conducted . both humans and laboratory mice exhibit detectable levels of il-15 in the circulation , ( for example , [ 9 , 10 , 15 , 25 ] ) , allowing for the possibility that il-15 can exert endocrine ( as well as paracrine ) effects on cell types which do not express il-15 itself . because of the high expression of il-15 in skeletal muscle and evidence that other cytokines such as il-6 are released from muscle following physical activity , some investigators have suggested il-15 functions as a myokine which exerts positive effects on body composition via an endocrine mechanism [ 1316 ] . conflicting evidence for il-15 as a myokine is reviewed below . as mentioned above , another tissue which exhibits high expression of both il-15 mrna and protein is placenta [ 17 , 26 , 27 ] . in a study comparing concentrations of numerous cytokines in amniotic fluid and sera of normal pregnant women , chow et al . reported significantly higher il-15 concentrations in serum than in amniotic fluid , consistent with literature indicating many cytokines do not cross the placental barrier . however , studies of whether gravid animals or pregnant women display increased circulating levels of il-15 compared to nongravid or nonpregnant females are lacking . in any case , such an observation would not apply to males and does not preclude skeletal muscle or other tissues as additional sources of il-15 in both genders . two il-15 mrna isoforms are generated from a single il15 gene in humans and mice [ 28 , 29 ] . the two isoforms differ in the lengths of the signal peptides and have been designated long signal peptide ( lsp ) and short signal peptide ( ssp)-il-15 mrna . however , tissue expression and intracellular trafficking of these isoforms are dissimilar [ 28 , 29 ] . ssp - il-15 mrna is highly transcribed in heart , and also expressed in thymus , testes , and appendix , whereas lsp - il-15 mrna is transcribed strongly in skeletal muscle and placenta , and at lower levels in heart , lung , liver , thymus , and kidney . ssp - il-15 does not appear to be secreted , and either functions intracellularly or is released following cell damage [ 28 , 29 ] . lsp - il-15 is secreted ; however , the unusually long 48 amino acid signal peptide renders il-15 secretion extremely inefficient [ 2831 ] . lsp - il-15 protein translation is impeded by multiple augs ( initiation codons ) in the 5 untranslated region [ 3133 ] . because of the inefficiency of il-15 translation and secretion , correlations between il-15 mrna levels and secretion of il-15 protein are often weak ( reviewed in [ 18 , 33 ] ) . a systematic comparison of il-15 protein expression among tissues has not been conducted , and it is technically difficult to demonstrate secretion from a specific tissue in vivo , particularly in mouse models . the mouse 3t3-l1 adipogenic cell line does not express levels of il-15 mrna detectable by highly sensitive real - time pcr at any stage of differentiation . however , another study demonstrated primary pig adipocyte cultures express il-15 mrna at low basal levels , which are upregulated following stimulation with interferon- . whether il-15 protein was produced or released into the culture medium differences between rodents and swine , and/or between adipogenic cell lines and primary cultures , as well as between adipocytes in vivo and in vitro , are quite possible . in obesity , adipose tissue develops an inflammatory environment due to infiltrating macrophages which are a source of numerous proinflammatory cytokines [ 34 , 35 ] . in a mouse strain highly susceptible to oxidative stress , high dietary calcium in conjunction with an obesigenic diet significantly stimulated il-15 mrna expression in both visceral fat and skeletal muscle tissue , but it was not determined which cell types were responsible for the upregulated il-15 message . a systematic study of il-15 mrna and protein expression in various depots of fat tissue , in different physiological conditions , has not been performed . thus , whether adipocytes and/or adipose tissue can express and secrete il-15 protein in basal conditions or in inflammatory challenges associated with obesity remains unclear . the il-15 gene ( il15 ; human accession number u14407 ) is mapped to human chromosome 4q31 and the central region of chromosome 8 in mice . a recent genome - wide survey of human copy number variations which correlated with obesity revealed a large ( 2.1 mb ) deletion of a region which included both il15 and the gene encoding the mitochondrial uncoupling protein ucp1 . since the role of ucp1 in modulating energy balance is well described , the potential contribution of loss of il15 by this large deletion was not additionally considered . however , research findings from adipogenic cell cultures , laboratory animals , and human subjects all suggest il-15 may also function as an antiobesigenic factor . in a study of human subjects comprising a wide range of body mass indices ( bmi ) , nielsen et al . found negative associations between plasma il-15 concentrations and bmi ( p < .001 ) , total fat mass ( p < .001 ) , trunk fat mass ( p < .01 ) , and limb fat mass ( p < .05 ) . negative associations between muscle il-15 mrna and obesity parameters were also observed in that study . a similar finding was reported by barra et al . who observed obese human subjects exhibited lower circulating il-15 levels than lean subjects . however , christiansen et al . reported decreased circulating il-15 concentrations following diet - induced weight loss in obese human subjects . two snps in human il15 ( rs1589241 and rs1057972 ) are associated with various predictors of the metabolic syndrome , bmi , and muscle strength [ 10 , 11 ] . these snps are located in the 5 and 3 untranslated regions ( utrs ) of the gene , suggesting they could modulate il-15 expression . mice with targeted deletion of il15 ( il-15ko mice ) exhibit higher amounts of body fat than control mice . conversely , transgenic mice which were engineered for elevated circulating levels of il-15 ( il-15 tg mice ) , expressed from a skeletal muscle - specific promoter , exhibited lower levels of body fat than closely related controls , and were resistant to diet - induced obesity . in the same study , mice which expressed high intramuscular levels of il-15 but which did not exhibit elevated serum il-15 levels showed no differences in adiposity compared to controls , suggesting il-15 must be secreted into the circulation to exert its effects on adipose tissue . since this is an artificially constructed system , it can model , but does not prove , the hypothesis that muscle - derived il-15 acts as a myokine in the native state . il-15 has also been introduced into wild - type laboratory rodents by injection of recombinant il-15 protein [ 13 , 40 ] , by adenoviral expression vectors , and by dna electrotransfer into skeletal muscle . in these studies , il-15 administration reduced fat mass by as much as 30% in normal rodents and 10% in obese rodents . importantly , inhibition of fat deposition was observed in the absence of an effect of il-15 on food consumption [ 13 , 40 ] . additionally , in the studies of both il-15ko mice and the gain - of - function il-15 tg mice described above [ 40 , 42 ] , no effects of loss or overexpression of il-15 on food intake were observed . therefore , the effects of il-15 on adipose tissue are not likely to be due to an indirect effect of modified energy intake . however , one report indicated chronic treatment of rats with il-15 slightly inhibited intestinal absorption of triaclglycerols specifically . lean body mass is unaffected by il-15 [ 13 , 40 , 42 ] , indicating il-15 does not induce a cachectic state ; indeed , recombinant il-15 injection can prevent loss of skeletal muscle mass in rodent models of cancer cachexia . il-15 injection inhibited fat deposition in both wild - type and leptin - deficient obese ( ob / ob ) mice . il-15 administration to lean rats also inhibited fat deposition , but it was unable to inhibit fat deposition in leptin receptor - deficient obese ( fa / fa ) zucker rats . obese , but not lean , rats exhibited significant decreases in adipose tissue expression of mrna for two of the subunits of the heterotrimeric il-15 receptor , the il-2 receptor beta and gamma subunits ( il-2r and il-2r ) , while expression of adipose tissue il-15r mrna was unchanged . this observation suggests that adipose tissue of obese zucker rats failed to respond to il-15 because the signaling subunits ( il-2r and il-2r ) of the il-15 receptor were downregulated in adipose tissue in this strain , and that the effect of il-15 on adipose tissue is direct . the direct effect of il-15 on adipose tissue was confirmed using adipogenic cell cultures derived from several mammalian species , including human [ 14 , 40 , 46 ] . recombinant il-15 administration inhibited preadipocyte differentiation and lipid deposition in the immortalized mouse 3t3-l1 cell line . moreover , in differentiated 3t3-l1 adipocytes , il-15 dose - dependently stimulated secretion of the insulin - sensitizing and antiobesigenic factor adiponectin . similar results were observed by another laboratory using cultured primary porcine adipocytes , in which il-15 potently stimulated lipolysis and modestly inhibited lipogenesis . finally , barra et al . found that administration of il-15 to lipoaspirate - derived human adipocyte cultures inhibited lipid deposition . the molecular pathways mediating the effects of il-15 on adipose tissue have not been characterized in detail . one study suggested il-15 upregulates expression of calcineurin mrna , a factor which inhibits adipocyte differentiation . in contrast to adipose tissue , il-15 mrna and protein expression has been observed consistently in skeletal muscle and skeletal muscle - derived culture systems [ 23 , 4851 ] , where it functions to modulate myofibrillar protein dynamics [ 23 , 44 ] . at the protein level , il-15 has been immunolocalized to human skeletal muscle fibers in tissue sections which contained few il-15-positive infiltrating cells . detected il-15 protein by immunohistochemistry and western blotting in human skeletal muscle with both type-1 and type-2 fiber dominance , which did not correlate with the respective levels of il-15 mrna expression in these muscles . expression of il-15 mrna and biologically active il-15 protein has been detected in primary human myogenic cultures , human rhabodomyosarcoma - derived cell lines , and a rat clonal myogenic cell line . il-15 mrna expression is low , but detectable , in mouse c2c12 skeletal myogenic cultures at the myoblast stage , but it is induced about 10-fold upon differentiation . therefore , published evidence indicates il-15 is expressed by skeletal muscle fibers themselves , not vascular , connective tissue , or lymphoid infiltrating cells present in muscle and in primary cultures . however , it is unclear if these observations translate to meaningful contributions to circulating il-15 levels by skeletal muscle tissue . several cytokines , most notably interleukins 6 , 8 , and 10 , are released from skeletal muscle following physical activity , resulting in large changes in plasma concentrations of such myokines [ 52 , 53 ] . one study found that in human skeletal muscle , il-15 was the mostly highly expressed of all cytokines measured at the mrna level . since both il-15 and physical exercise have positive effects on body composition , it is reasonable to hypothesize il-15 release following exercise may mediate some of the beneficial effects of physical activity [ 16 , 54 ] . however , there are conflicting reports as to whether physical activity affects il-15 expression in skeletal muscle and/or increases circulating il-15 levels . in a study of young untrained and 10-week - trained human subjects , riechman et al . demonstrated an increase in plasma il-15 protein levels immediately following whole - body resistance exercise and speculated that il-15 was released following exercise via microtears in muscle fibers . however , there was no effect of training on the postexercise release of il-15 in that study , casting doubt upon muscle damage as the mechanism . in contrast , a similar study using untrained human subjects reported no increase in muscle or circulating il-15 protein levels at intervals from 6 to 48 hours following an intensive resistance exercise protocol which involved only the quadriceps muscles ; however , increases in quadriceps muscle il-15 mrna expression were observed 24 hours after exercise . discrepancies between these studies may be due to the differences in the intervals between exercise and blood sampling . prior training or physical condition may modulate the purported release of il-15 from skeletal muscle tissue by exercise . using strength - trained human subjects , nieman et al . similarly , ostrowski et al . observed no changes in plasma il-15 following 2 h of treadmill running by 2 male athletes , and andersson et al . found no acute effects of competition on il-15 levels in elite female soccer athletes . in contrast , using sedentary postmenopausal women , prestes et al . reported an increase in plasma il-15 levels 48 hours following the first session of resistance training but no effect following 16 weeks of training . finally , christiansen et al . studied chronic ( not acute ) plasma cytokine levels in obese , physically inactive subjects subjected to a program of intensive ( hypocaloric ) dietary intervention , aerobic exercise , or the combination of diet and exercise . compared to baseline , diet alone and the combination of diet and exercise significantly decreased circulating il-15 levels , whereas aerobic exercise alone had no effect . however , given the extremely hypocaloric nature of the dietary intervention ( 600800 kcal / day ) , it is possible some loss of muscle tissue occurred , which was not examined similarly reported no effect of a 12-month program of combined aerobic , strength and balance intervention on circulating il-15 levels in frail elderly subjects . it is possible that differences among these studies were due to the use of highly trained versus untrained , obese , and/or elderly subjects and/or to the difference between aerobic versus resistance exercise . therefore , evidence for the hypothesis that il-15 is released following exercise is mixed and largely negative . moreover , an increase in circulating il-15 levels after exercise is only circumstantial evidence of its derivation from skeletal muscle tissue . for example , although skeletal muscle expresses the myostatin - inhibiting factor follistatin , a recent study provided evidence that the postexercise rise in circulating follistatin is mostly likely derived from the liver rather than from muscle tissue . the concept of il-15 as a myokine does not necessarily imply that it is released by exercise , rather it implies simply that the factor is derived from skeletal muscle tissue and is released in quantities sufficient to have an endocrine effect on other cell types . such release also does not necessarily imply that expression of the purported myokine is specific to skeletal muscle tissue . on the other hand , skeletal muscle tissue comprises almost 50% of body mass and is specialized for protein synthesis , if not secretion , so a small release of a bioactive factor from each muscle could have a large systemic effect . sporadic reports of other inflammatory , hormonal , or nutritional factors which affect muscle il-15 mrna expression and circulating il-15 protein levels have been published . administered the synthetic progestin megestrol acetate at 800 mg / d for 12 wk , with or without testosterone ( 100 mg / wk ) , resistance training , or the combination of resistance training and testosterone . progestin ingestion , but no other treatment , caused highly significant increases in circulating il-15 levels , but this treatment did not correlate with changes in muscle mass or body composition . as mentioned above , dietary calcium significantly stimulated il-15 mrna expression in murine skeletal muscle tissue ( as well as visceral fat ) in obesigenic conditions and oxidative stress , which was interpreted as an increase in anti - inflammatory cytokine expression due to calcium - mediated inhibition of 1,25-dihydroxyvitamin d3 . in myogenic cell cultures , overexpression of an orphan nuclear hormone receptor which is highly expressed in muscle , retinoid - related orphan receptor gamma , upregulated both il-15 and myogenin mrna , as well as expression of several genes which regulate lipid and carbohydrate metabolism , insulin sensitivity , and reactive oxygen species . cultured rat myocytes upregulated il-15 mrna in response to both the inflammatory factor interferon- and the anti - inflammatory cytokine il-4 , while primary human myoblast cultures increased expression of both intracellular and secreted il-15 protein in response to several inflammatory mediators ( interferon- , interleukin-1 , interleukin-1 , tnf- , and lipopolysaccharide ) . these observations suggest muscle il-15 expression is modulated by dietary , hormonal , and inflammatory status , but , as in the experiments dealing with exercise , a clear understanding of the regulation of il-15 expression by such factors in skeletal muscle tissue is lacking . increased age is an important predictor of obesity and the metabolic syndrome [ 3 , 62 ] . reports in animal models on the effects of age on il-15 expression in muscle and in serum are conflicting . . found that il-15 mrna was elevated in both slow and fast aging rat muscles compared to young muscles . the same study found that il-15 mrna was elevated in atrophied slow soleus muscles of young rats but not in the fast plantaris muscle . a similar effect of aging and immobilization recovery on il-15 mrna in rat muscles was reported in another study . however , given the complex regulation of il-15 described above , it is unclear whether these changes in il-15 mrna expression reflect similar changes in muscle il-15 protein expression and secretion in these physiological states . indeed , in mice , both muscle and serum il-15 protein levels were reported to decline progressively with increasing age ; however , this decline was not accompanied by decreases in transcription of either ssp- or lsp - il-15 mrna . rather , while expression of the membrane - associated form of il-15r ( mbil-15r ) did not change with age , an age - related decline in expression of muscle soluble il-15r ( sil-15r ) mrna in muscle tissue was demonstrated , suggesting decreased expression of this factor ( reviewed below ) could lead to age - related declines in il-15 secretion from muscle . in another study , a similar decline in muscle il-15 protein levels ( and muscle il-15r mrna expression ) in ad lib - fed rats was described , but no declines in il-15 protein levels or il-15r mrna expression were observed in long - lived , calorie - restricted rats . intriguingly , gangemi et al . observed that while serum il-15 concentrations exhibited a downward trend with age in unselected human subjects , individuals with unusually long lifespans ( 95 to more than 100 years ) and still living independently had significantly elevated circulating il-15 levels , suggesting elevated il-15 levels conferred some protection against age - related illness . il-15 signaling is transduced either through a heterodimeric receptor comprising il-2r and il-2r , or through a heterotrimeric receptor comprising mbil-15r plus il-2r and il-2r . the il-2r and il-2r subunits are responsible for signal transduction , while mbil-15r has only a short cytoplasmic region and functions primarily to confer high - affinity binding to the receptor complex . cells can express heterodimeric and heterotrimeric il-15 receptor complexes simultaneously or express il-15r in the absence of the other two subunits . unexpectedly , mice in which the il-15r is deleted ( il15rko mice ) are leaner , rather than fatter , than controls . this observation suggests a complex role for this receptor subunit in control of body fat , and is consistent with the idea that the il-15r can function in roles other than a membrane - bound receptor component . il15ra ( human accession number u31628 ) is located on mouse chromosome 2 and human chromosome 10p . loci on human chromosome 10p have been strongly linked to both obesity and type-2 diabetes [ 70 , 71 ] . human and mouse il15ra exhibit similar genomic structures ( [ 67 , 69 ] and reviewed in ) . il15ra has an efficient signal sequence ( exon 1 ) , a ligand - binding domain ( exon 2 ) , a transmembrane domain ( exon 6 ) , and , as mentioned , a fairly short cytoplasmic domain ( exon 7 ) . exon 2 encodes a conserved protein - binding region known as a sushi domain , which is responsible for the binding affinity of il-15r for the il-15 ligand [ 67 , 69 ] . a number of il-15r mrna splice variants have been reported in both mouse and human , some of which encode soluble forms which may modulate il-15 secretion and bioavailability . because both isoforms of il-15 have inefficient signal sequences , recent experimental evidence suggests il-15 is brought to the cell surface by intracellular association with either sil-15r or mbil-15r , both of which would include the efficient exon-1-encoded il-15r signal sequence . one , which arises by differential splicing , comprises only exons 1 and 2 , so does not contain a transmembrane domain . il-15 secretion as an il-15/sil-15 r complex is thereby facilitated following intracellular binding of il-15 and this truncated receptor isoform [ 8 , 9 ] . another form of sil-15r arises by proteolytic cleavage of mbil-15r ( which can be complexed to il-15 ) by the matrix metalloproteinase tnf- converting enzyme ( tace ) . release of this form of circulating il-15/sil-15r complex is dependent upon tace activity , which in turn is correlated with obesity and insulin resistance in mice and humans [ 73 , 74 ] . uncomplexed il-15 and the two types of il-15/sil-15r complexes appear to be in a dynamic equilibrium in the circulation , and the two soluble receptor variants can compete for binding of il-15 . however , while both forms facilitate il-15 secretion and can increase il-15 half - life in the circulation , the two variants have differential effects on il-15 bioactivity ; additionally , the two types of il-15/sil-15r complexes can differentially bind heterodimeric versus heterotrimeric il-15 receptors [ 8 , 9 ] . therefore , regulation of the various forms of sil15r may be an important element modulating il-15 secretion and bioactivity . in this regard , human genetic studies by three separate laboratories have identified several single - nucleotide polymorphisms ( snps ) in il15ra which impact muscularity , fat deposition , and markers of the metabolic syndrome [ 13 , 14 , 19 ] . for example , a snp ( rs2228059 ) in exon 3 correlates with serum triglyceride levels in males . an a to g variation in the exon 5/intron border ( snp accession rs3136618 ) is associated with the so - called normal weight obese / de lorenzo syndrome , characterizing young women with normal bmis but greater than 30% body fat and the presence of several markers of metabolic syndrome . in a separate study of male subjects by a different group , this same snp correlated with muscularity . whether il-15 is differentially complexed with sil-15r variants during obesity has not been studied , but the existence of multiple snps in il15ra which correlate with body composition suggest this is a fertile area for future investigation . il-15 and il-15 receptor subunits are expressed in several regions of the cns [ 68 , 75 , 76 ] . il-15 has been reported to modulate non - rem sleep and to play a protective role in neuroinflammation . recently , il15rko mice were reported to be hyperphagic and hyperactive , with the suggestion that il-15 may control hypothalamic pathways which regulate energy balance . however , il-15 expression , trafficking and effects in the cns are an additional , relatively unexplored area of investigation which could impact the balance of energy expenditure and energy intake . findings from cell culture , laboratory animal , and human studies indicate il-15 regulates adipose tissue deposition by a direct mechanism . there are conflicting reports concerning the tissue origin of circulating il-15 and whether it can act as a circulating , muscle - derived whatever the tissue of origin , recent evidence supports the hypothesis that circulating il-15 levels and il-15 bioactivity are determined by differential association of il-15 with sil-15r variants . the concordance of basic science findings and human genetic studies suggests the sil-15r , which in turn regulates il-15 , is an important factor influencing body composition and insulin sensitivity . therefore , further investigation into the complex il-15/il-15r axis is needed to determine if this system can be exploited for regulation of adiposity .

interleukin-15 ( il-15 ) is a cytokine which inhibits lipid deposition in cultured adipocytes and decreases adipose tissue deposition in laboratory rodents . in human subjects , negative correlations between circulating il-15 levels and both total and abdominal fat have been demonstrated . deletions of il15 in humans and mice are associated with obesity , while gain - of - function il-15 overexpressing mice are resistant to diet - induced obesity . il-15 is highly ( but not exclusively ) expressed at the mrna level in skeletal muscle tissue , and the regulation of il-15 translation and secretion is complex . conflicting evidence exists concerning whether circulating il-15 is released from skeletal muscle tissue in response to exercise or other physiological stimuli . the il-15 receptor - alpha ( il-15r ) subunit has a complex biochemistry , encoding both membrane - bound and soluble forms which can modulate il-15 secretion and bioactivity . the gene encoding this receptor , il15ra , resides on human chromosome 10p , a location linked to obesity and type-2 diabetes . several single - nucleotide polymorphisms ( snps ) in human il15ra and il15 correlate with adiposity and markers of the metabolic syndrome . genetic variation in il15ra may modulate il-15 bioavailability , which in turn regulates adiposity . thus , il-15 and the il-15r may be novel targets for pharmacologic control of obesity in the human population .

1. Introduction 2. Molecular Genetics and Expression of IL-15 with Regard to Adipose Tissue 3. Effects of Interleukin-15 on Adipose Tissue 4. IL-15 as a Potential Myokine 5. The Soluble IL-15 Receptor-Alpha as a Modulator of IL-15 Secretion 6. Effects of IL-15 via the Central Nervous System (CNS) 7. Conclusions

PMC4755995

neuromuscular control can be defined as an interaction between the nervous and musculoskeletal systems to produce a desired effect or response to a stimulus3 . enhancements of several factors such as muscular strength , power , and endurance are directly related to the improvement in an athletes exercise performance . recently , various kinds of training methods have been implemented in order to improve the muscular power of sports players ; plyometric training has been a typical intervention for enhancing the muscular function4 , power5 , and running performance ability6 . however , it might cause musculoskeletal injuries at landing phase7 , 8 . in this sense , whole - body vibration exercise ( wbve ) , regarded as a neuromuscular training method utilizing a moderate vibration stimulus to improve muscular strength and power9,10,11,12 , has become a practical exercise intervention for athletes . the potential factors expected from wbve are vibration type , frequency , body position , and additional load13 . among these , a change in the body position through different knee bending angles14 , 15 might be one of the most crucial factors that can augment muscular function with wbve . however , previous studies have not suggested the optimal squatting position that can maximize the effect of wbve , and explained the interrelationship between the optimum knee angle during a squat and wbve . accordingly , the primary objective of this study was to validate the optimal squatting position that maximizes muscular function when wbve is performed with a certain vibration frequency , and for a certain time duration . 12 subjects ( men , aged 26 1.9 years , height 175.91 7.3 cm , weight 74.4 10.88 kg ) who did not exercise regularly participated in this study . the participants were explained the procedures and the purpose of the study , and were requested to submit a written consent form . this study was approved by the university of sunmoon research ethics review committee ( syuirb 2010 - 007 , 27 february 2015 ) according to the declaration of helsinki . the protocol of this study for assessing the vibration frequency and time is based on a previous study conducted by bosco14 , and interventions for the different squatting positions are adopted from studies conducted by deleclus15 and jacobs16 . in addition , this study defined the squatting position according to the knee and hip angle as lower squatting position ( ls ) ( 90 ) , middle squatting position ( ms ) ( 140 ) , and high squatting position ( hs ) ( 170 ) . wbve was performed at 26 hz frequency with 5 sets of exercise in one minute . after resting for 6 minutes , another session of 5 sets all participants implemented the exercise program without wearing shoes and socks in order to eliminate additional effects like cushion of the shoes . moreover , all measurements in this study were performed by the same tester , and the measurement schedules were planned with at least one - week interval after a certain session to minimize the learning effect . data from knee isokinetic muscular function test were evaluated with an isokinetic dynamometer , bte primusrs system ( bte technologies inc . , usa ) , and jump performance variables were measured with g - jump ( bts bioengineering , italy ) . wbve equipment ( galileo 2000 , novotec medical , germany ) was used to determine the effects of the exercise program . statistical analysis was performed using spss / pc 18.0 statistical program for mean and standard deviations . analysis of variance ( anova ) was performed for repeated measurements to analyze the differences in knee isokinetic muscular function and jump performance depending on the squatting position . moreover , the post - hoc test with least significance difference ( lsd ) was used in this study . statistical significance level ( ) for data analysis was set at p < 0.05 . in table 1table 1.differences in isokinetic muscular function of the knee and jump performance according to the squatting positionpre - interventionhs ( 170)ms ( 125)ls ( 90)comparison60/sec ( nm)extension518.3 68.9542.2 56.3537.0 5.5550.3 67.8a < dflexion339.0 50.5392.2 49.9387.8 47.8412.3 61.9a < b , c , d180/s total work(j)2,188.7 346.62,728.0 478.22,787.0 444.02,685.9 432.9a < b , c , djump height(cm)49.16 2.850.59 3.451.04 3.7651.32 3.6a s : second . * a significant difference between the interventions ( p < 0.05 ) . a : pre - intervention , b : hs , c : ms , d : ls , the difference in isokinetic muscular sunction of the knee and jump performance depending on the squatting position , and the knee angles for each squatting position ( hs , ms , and ls ) are represented . significant difference was seen in the knee flexor strength at 60/s and knee muscle endurance at 180/s after wbve intervention in all squatting positions ( p < 0.05 ) . for the knee extensor strength at 60/sec , among the squatting positions significant difference in addition , jump height was significantly increased in all squatting positions ( p < 0.05 ) . all variables are mean standard deviaion . s : second . * a significant difference between the interventions ( p < 0.05 ) . a : pre - intervention , b : hs , c : ms , d : ls in this study , it can be said that wbve might have provided an external stimulus for improving muscular strength , power , and performance for sports players during the time period . the present study shows that isokinetic muscular functions such as muscular strength and endurance had significantly increased in all squatting positions with wbve when compared to pre - exercise state . this implies that primary endings of the muscle spindle are stimulated by the vibrations , which excites the motor neurons causing contraction of motor units , resulting in tonic contraction of the muscle known as tonic vibration reflex15 , 17 , and greater training effects are stimulated by increased motor unit recruitment as an effect of the tonic vibration reflex18 . it is thought that the results of this study demonstrate the improvement in muscular functions by increasing the co - contraction ability of the synergist muscles . in addition , it is noteworthy that the muscular function in ls ( 90 knee angle ) was significantly better than the pre - intervention state . although no significant difference was found among the squatting positions , ls with wbve might be able to maximize the neuromuscular exercise effect in terms of amplifying the sensitivity of the muscle spindle . it can be said that the stimulated spindle of vastus medialis and lateralis and its muscular power are the optimum results of wbve with ls . these aspects could increase the tonic vibration reflex , and , as a result , the exercise ability such as jump performance was better in ls in comparison with other squatting positions ( ms and hs ) . in other words , when applying wbve with squatting position , the knee angle is one of the most important factors when it comes to considering the optimal exercise effect . more importantly , wbve might be able to offer positive training effects to the sports players hoping to improve their muscular power and endurance within a short period , as shown in this study , when time or exercise methods are lacking . in conclusion , this study suggests that wbve at 26 hz vibration frequency with 90 angle of hip and knee might be a feasible exercise intervention for sports players ; however , other combinations between the knee angle and vibration frequency for an optimal exercise effect should be considered in future studies .

[ purpose ] the purpose of this study was to investigate the effects of whole - body vibration exercise ( wbve ) on isokinetic muscular function of the knee and jump performance depending on different squatting positions . [ subjects ] the subjects were 12 healthy adult men who did not exercise regularly between the ages of 27 and 34 . [ methods ] wbve was performed with high squat position ( sp ) , middle sp , and low sp . before and after the intervention , isokinetic muscular function of the knees and jump performance were measured . [ results ] knee flexion peak torque at 60/s and total work at 180/s were significantly increased after implementing wbve . jump height also significantly increased after completing the exercise at all positions in comparison with the pre - exercise programs . [ conclusion ] the results of this study suggest that sp during wbve is an important factor stimulating positive effects on muscular function .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC3399426

as the predominant cause of liver injury , alcoholic liver disease ( ald ) remains a major cause of both morbidity and mortality in the western world . alcohol consumption in developing countries is increasing significantly and ald is becoming a major socioeconomic burden . in 2003 , it was estimated that alcohol was responsible for 44% of all deaths from liver disease . despite its overwhelming prevalence , only 1015% of alcoholics ever develop hepatitis and cirrhosis . as a result , it is crucial to intervene therapeutically during the early initiating stages of the disease where recovery is possible . ald is a multifactorial disease , resulting from numerous cellular derangements , creating difficulties when trying to develop a targeted therapy . one of the major confounding factors in our understanding of the pathogenesis of ald is the generation of reactive oxygen species ( ros ) resulting from the oxidative metabolism of ethanol . a well - documented consequence of ethanol ingestion is the induction of cytochrome p450s , specifically cyp2e1 , and depletion of cellular antioxidants leading to a prooxidative state . while enhanced generation of ros is likely to occur through multiple mechanisms , cyp2e1 and nadph oxidase ( nox ) are thought to be major sources [ 2 , 4 , 5 ] . sustained exposure to ros leads to prolonged oxidative stress , which promotes lipid peroxidation and generation of reactive aldehydes , such as 4-hydroxy-2-nonenal ( 4-hne ) . this highly reactive aldehyde has been shown to covalently modify numerous cellular proteins , often resulting in alterations in protein structure , localization , function , and/or activity . protein folding is a critical function of the hepatic er , yet despite the presence of a large number of chaperone proteins in the er , an estimated 30% of nascent proteins fail to reach their properly folded state [ 7 , 8 ] . given this high frequency of improper folding , the er incorporates a quality control system known as er - associated degradation ( erad ) , which eliminates unwanted proteins through ubiquitination and subsequent proteasomal degradation . under situations of sustained stress , where erad processes can not eliminate all unfolded / misfolded proteins , the cell undergoes a series of adaptive signaling events termed the unfolded protein response ( upr ) . the overall goal of upr signaling is to selectively increase the transcription of chaperones and increase erad processes , while decreasing overall translation . upr signaling is also known to induce selective signaling associated with both lipid and cholesterol homeostasis through activation of the sterol regulatory element - binding proteins ( srebps ) [ 11 , 12 ] . these transcription factors tightly regulate fatty acid synthesis ( srebp-1 ) and cholesterol synthesis ( srebp-2 ) . have implicated a role for the upr in a rodent model of ald [ 13 , 14 ] . although the proposed mechanism of this ald - associated upr induction is through increased plasma homocysteine , others have suggested a prominent role for oxidative stress in the activation of the upr [ 8 , 1517 ] . in addition , sustained upr activation has been shown to lead to enhanced ros generation resulting in oxidative stress through activation of the er oxidoreductases ( ero1 ) and nad(p)h oxidase 4 ( nox4 ) [ 17 , 18 ] . altered redox control in the er is linked to numerous hepatic disease states , including ald [ 15 , 19 ] . the transition from steatosis to steatohepatitis is considered to be the critical pathogenic determinant in ald , furthering a need for therapeutic intervention during the early initiating steatotic stages of disease progression . the mechanisms behind this transition remain elusive , and research on ald is rarely focused on this stage . in this paper , we demonstrate a clear role for oxidative stress in the initiation of ald that precedes the involvement of the upr . these findings show , for the first time , a clear dissociation between oxidative stress and upr signaling in the initiation of ald . all procedures involving animals were approved by the institutional animal care and use committee of the university of colorado and were performed in accordance with published national institutes of health guidelines . male c57/bl6j mice ( 12 per group ) were utilized for the analysis and characterization of ethanol - mediated liver damage . briefly , mice were fed a modified lieber - decarli liquid - based diet ( bio - serv , frenchtown , nj ) for a period of 6 wk . the diet consisted of 44% fat - derived calories and 16% protein - derived calories , and the remaining balance was comprised of either maltose - dextrin or ethanol - derived calories . ethanol - fed mice began the study on a diet consisting of 2% ( v / v ) ethanol , with the ethanol - derived calories increasing on a weekly basis until sacrifice ; week 6 consisted of 6% ethanol ( v / v ) or 31.8% ethanol - derived calories . pair - fed control animals received the remaining caloric intake from carbohydrate source ( the caloric content of the diet used for the ethanol administration paradigm is presented in table 1 ) . food consumption was monitored daily , and body weights were measured once per week . upon completion of the study , animals were anesthetized via intraperitoneal injection with sodium pentobarbital and euthanized by exsanguination . blood was collected from the inferior vena cava , and plasma was separated through centrifugation and assayed for alanine aminotransferase ( alt ) activity ( diagnostic chemicals limited , oxford , ct ) . livers were excised , weighed , and frozen for biochemical characterization or subjected to differential centrifugation for subcellular fractionation as previously described . as a positive control , endoplasmic reticulum stress was induced in c57bl/6j mice ( 711 wks ) by a single ip injection with tunicamycin ( tm ) at 0.5 mg / kg body weight . liver triglycerides were measured using a 2 : 1 chloroform : methanol extraction from liver homogenates using a kit from diagnostic chemicals limited . protein concentrations were determined using a bca protein assay from pierce ( rockford , il ) or a modified lowry protein assay from bio - rad ( hercules , ca ) . thiobarbituric acid reactive substances ( tbarss ) were measured in whole liver homogenates ( 500 g ) by incubating samples in thiobarbituric acid reagent ( 15% w / v trichloroacetic acid , 0.375% w / v thiobarbituric acid , and 0.25 n hcl ) . samples were boiled at 100c for 15 min , cooled to room temperature , and then centrifuged at 10,000 rpm . absorbance of the cleared supernatant was read spectrophotometrically on a microplate reader at 535 nm , and the concentration of malondialdehyde ( mda ) was calculated from the extinction coefficient , = 156,000 m cm . the activity of glutathione reductase ( gr ) was determined in cytosolic fractions according to babu et al . glutathione s - transferase ( gst ) activity was measured in whole liver homogenates according to rinaldi et al . . glutathione peroxidase ( gpx ) activity was determined in whole liver homogenates using a commercially available kit ( cayman chemical , ann arbor , mi ) . the enzymatic activity of glutaredoxin ( grx ) was determined using the heds assay according to mesecke et al . . briefly , blood was collected on a weekly basis from the submandibular area of the mouse and placed in a heparinized tube ; 0.6 m perchloric acid was added to each tube to precipitate protein . samples were then centrifuged at 20,000 g for 10 minutes , and isopropanol was added to each tube as the internal standard . samples were capped with air - tight rubber septums and heated for 15 min at 60c . head space gas was withdrawn ( 0.2 ml ) and injected with a gas - tight syringe into a hewlett - packard 5710a gas chromatograph ( injection port : 150c , detector : 250c , oven : 85c ) . separation of ethanol and isopropanol was performed on a 6 ft column packed with 60/80 carbopack b ( supelco , inc . , nitrogen was used as the carrier gas ( flow rate = 40 ml / min ) . ethanol concentrations were quantified against a standard curve using isopropanol as an internal standard to control for injection volume variation . mouse livers were homogenized in buffer containing 100 mm potassium phosphate , ph 7.4 , 1 mm edta , and 1 : 50 ( v / v ) protease inhibitor cocktail ( sigma aldrich ) . the homogenate was subsequently centrifuged at 4c at 20,000 g for 20 min . protein concentration of the crude extract was determined by the bradford method using bovine serum albumin as a standard . cbs activity assays were carried out using modifications to the method of kashiwamata and greenberg with a ninhydrin reagent specifically targeting cystathionine as described by villanueva et al . [ 27 , 28 ] . briefly , 700 g of crude liver extract protein was incubated in assay buffer containing 100 mm tris - hcl , ph 8.6 , 10 mm l - serine , 1 mm pyridoxal 5-phosphate , and 2 mm dl - propargylglycine at 37c for 5 min . l - homocysteine was then added to a final concentration of 26 mm and a final volume of 50 l to start the reaction at 37c for 1 h. the reaction was stopped by the addition of 50 l of 10% trichloroacetic acid ( tca ) . samples were subsequently centrifuged at 20,000 g for 5 min , and a 50 l sample of the supernatant was taken for cystathionine quantification as described previously . duplicate reactions were run in the absence of 10 mm l - serine as a negative control for each sample . cgl activity assays were performed using an enzyme - coupled assay with lactic dehydrogenase ( ldh ) as described previously . assay reactions were preincubated at 37c and contained 100 mm potassium phosphate , ph 8.0 , 2 mm l - cystathionine , 175 mm nadh , and five units of ldh . reactions were started by the addition of 25 g of crude liver extract to give a final reaction volume of 100 l . the oxidation rate of nadh was monitored at 340 nm for 2 min at 37c as an index of cgl activity . gcl activity was assayed from 1.0 mg of whole liver extract utilizing previously described protocols . briefly , fresh liver tissue was homogenized in 0.1 n perchloric acid using a sonicator and centrifuged at 13,000 rpm for 15 min , and the cleared supernatant was further neutralized with sodium bicarbonate and treated with 1 : 10 iodoacetic acid ( iaa ) ( 14.9 mg / ml ) in the dark for 45 min at room temperature . samples were then derivatized ( 1 : 1 ) with sanger 's reagent ( 1.5% 1-fluoro-2,4-dinitrobenzene ( dfnb ) in absolute ethanol ) overnight in the dark at room temperature . samples were applied ( 50 l injection ) to a supelco lc - nh2 5 m , 25 cm 4.1 mm column ( sigma aldrich , st . solvent a ( 80% methanol ) and solvent b ( 80% solvent b , 20% acetic acid / ammonium acetate solution ( 756 ml glacial acetic acid , 244 ml water , and 308 g of ammonium acetate ) ) were used for the separation of derivatized reduced ( gsh ) and oxidized ( gssg ) glutathione using a shimadzu lc-10ad dual pump system ( flow rate of 1.0 ml / min ) coupled to a shimadzu spd - m10av diode array detector ( kyoto , japan ) set to 350 nm . hplc running conditions were as follows : isocratic period of 50% solvent b for 10 min , followed by a linear gradient from 50% solvent b to 95% solvent b for 15 min , for a total run time of 35 min . typical elution times were 11.55 min for gsh and 17.05 min for gssg . nanomolar concentrations of gsh and gssg were calculated against a standard curve of 7 points for each molecule . proteins from either whole liver extracts or subcellular fractions were subjected to standard sds - page or native page . native page was employed to analyze the relative levels of monomeric gclc and gcl holoenzyme . native samples were mixed with loading buffer lacking 2-mercaptoethanol and were not boiled prior to loading on 8% polyacrylamide gels without a stacking gel or sds . all gels were transferred to a hybond - p membrane ( ge healthcare , buckinghamshire , uk ) and then blocked for 30 min with a tris - buffered saline solution containing 1% tween-20 ( tbst ) and 5% nonfat dry milk ( nfdm ) . membranes were probed with primary antibodies directed against inositol - requiring enzyme 1 ( ire-1 ) and its phosphorylated form ( p - ire1 ) , eukaryotic translation initiating factor 2 ( eif2 ) , growth - arrest - and - dna - damage - inducible gene 153 ( gadd153 , also known as chop ) , srebp1 , srebp2 , nox-4 , lamin b1 , activating transcription factor 6 ( atf6 ) ( abcam , cambridge , ma ) , kdel ( grp78 and grp94 ) ( stressgen , ann arbor , mi ) , ero1 and ero1 ( proteintech group , chicago , il ) , p - eif2 ( epitomics , burlingame , ca ) , -actin ( sigma ) , and gclc and gclm ( antibodies were generously provided by dr . a horseradish peroxidase conjugated secondary ( jackson labs , bar harbor , me ) was then applied and membranes were developed using ecl - plus reagent from ge healthcare . chemiluminescence was visualized using a storm 860 scanner from molecular dynamics ( sunnyvale , ca ) . following excision , livers were sectioned and placed in 10% neutral buffered formalin for 8 h. samples were processed , imbedded in paraffin , and mounted on slides by colorado histoprep ( fort collins , co ) . one pair of slides was stained with hematoxylin and eosin ( h&e ) for histological characterization while the remaining slides were subject to deparaffinization and rehydration for immunohistochemical characterization using either custom antibodies generated in our laboratory directed against 4-hne modified proteins , anti - cyp2e1 ( calbiochem , san diego , ca ) , or anti - protein - ssg ( virogen , watertown , ma ) . liver samples from control and ethanol - treated mice were homogenized in 0.1 m tris - hcl and 0.25 m sucrose . homogenates were centrifuged at 105,000 g for 1 h , and the resulting supernatant ( cytosolic fraction ) was assayed for chymotrypsin - like activity utilizing a commercially available kit ( biovision , san francisco , ca ) . values were calculated against a standard curve and are reported as a % of control - fed mice . images of h&e or immunohistochemically stained liver sections were captured on an olympus bx51 microscope equipped with a four - megapixel macrofire digital camera ( optronics , goleta , ca ) using the pictureframe application 2.3 ( optronics , goleta , ca ) . statistical analysis and generation of graphs were performed using graphpad prism 4.02 ( graphpad software , san diego , ca ) . differences between control and ethanol - fed mice were assessed using a paired student 's t - test . as shown in table 1 , compared to their isocaloric controls , ethanol - fed mice displayed a decrease in overall body weight at each time point with the ethanol - treated mice weighing 16% less ( p < 0.001 ) at the conclusion of the study ( 6 weeks ) . as outlined , the ethanol concentration was increased incrementally throughout the course of the study . blood ethanol concentration ( bec ) was monitored and displayed a consistent increase at each time point , with maximum concentrations observed at week 6 ( 245.383 mg / dl 33.747 ) . consistent with early - stage ald , a significant increase in liver / body weight ratio was observed ( table 2 ) . this phenomenon was observed as early as 3 weeks , with the largest difference observed following 6 weeks . to assess liver damage , plasma alt values were determined at each time point ; ethanol - fed mice displayed greater than a two - fold increase in plasma alt levels by week 6 , with no significant difference being observed at weeks 1 and 3 . given these parameters , it was determined that mice treated for 6 weeks with ethanol displayed the earliest significant signs of ethanol - induced liver injury , demonstrating pathologies consistent with early - stage ald . hepatic steatosis is among the most prevalent and predictable outcomes of chronic ethanol consumption . as shown in figure 1(a ) , an increase in lipid was observed histologically as early as week 3 ( here seen as very small lipid vesicles in zone 2 ) , with a marked accumulation in lipid seen at week 6 ( here involving both zones 1 and 2 ) . triglyceride content was also quantified at each time point ; as shown in figure 1(b ) , only the week 6 ethanol - fed - mice displayed a significant increase in hepatic triglycerides . the increased accumulation of lipid observed in ald provides the ideal environment for the generation of lipid aldehydes resulting from oxidative stress [ 32 , 33 ] . as shown in figure 1(c ) , immunohistochemical analysis reveals a substantial increase in hepatic staining of 4-hne - modified proteins in as little as 3 weeks of feeding . the largest difference in staining , however , appeared following 6 weeks of ethanol ingestion with heavily stained hepatocytes localized predominately to zones 1 and 2 . to validate this observation , agreeing with the immunohistochemical staining in figure 1(c ) , a significant increase in tbars was observed at the week 6 time point , displaying nearly a two - fold increase . collectively , these parameters indicate pathologies consistent with early - stage ald , demonstrating a significant increase in hepatic lipid content and oxidative stress indices . as noted , decreased antioxidant capacity has been proposed to play a major role in the unbalanced generation of reactive aldehydes during ald . in line with this proposed mechanism , we sought to investigate the effects of ethanol consumption on hepatic gsh content and biosynthesis . as shown in table 3 , a significant depletion in reduced gsh was observed in mice treated for 6 weeks with ethanol compared to their respective isocaloric controls , indicating a decrease in the total antioxidant capacity of the liver at this time point . oxidized gsh ( gssg ) was also measured , observing no significant alterations at all time points analyzed . the reduction of gssg to gsh is catalyzed by glutathione reductase ( gr ) and is thought to be the predominant mechanism for gsh cycling . as shown in table 3 , gr activity was found to be significantly increased at the week 6 time point , with no variation observed at week 1 or week 3 . this phenomenon has been observed in other models for oxidative stress and has been proposed to be a compensatory mechanism . we and others have shown that oxidative stress and products of lipid peroxidation can also alter gsh biosynthesis via actions on gcl . this has been shown to occur through posttranslational modification of preexisting protein and/or changes in the relative levels of gcl holoenzyme , which has a significantly greater specific activity than monomeric gclc [ 30 , 35 ] . as shown in table 3 , the activity of gcl was significantly increased following 1 and 6 weeks of ethanol consumption . standard immunoblotting revealed that ethanol ingestion had no significant effect on gclc or gclm expression ( data not shown ) . analysis of gcl holoenzyme formation by native gel electrophoresis and immunoblotting revealed no significant effect of ethanol ingestion on the relative levels of gcl holoenzyme compared to monomeric gclc ( data not shown ) . while these findings suggest that ethanol - induced oxidative stress increases hepatic gcl activity , it should be noted that these gcl activity measurements were performed under saturating substrate concentrations and thus reflect optimal gcl enzymatic activity . importantly , such measurements do not take into account the potential contribution of altered substrate availability . indeed , cysteine availability is often the rate - limiting factor in gsh biosynthesis . to assess endogenous cysteine synthesis and its effects on gsh biosynthesis , the transsulfuration pathway was investigated , specifically cystathionine beta synthase ( cbs ) . as shown in table 3 , a significant decrease in cbs activity was observed at the week 6 time point ( p < 0.001 ) . this is likely a result of a significant decrease in protein expression ( ~30% , p < 0.05 , data not shown ) as a result of ethanol ingestion . the activity and expression of the transsulfuration enzyme cystathionine -lyase was also assessed and displayed no significant alterations in either parameter . collectively , these data suggest that not only alcohol consumption leads to the increased utilization of gsh as an antioxidant but also the endogenous synthesis of the gsh precursor cysteine is significantly altered , providing a dual mechanism for the observed decrease in gsh . the binding of gssg to protein thiols is referred to as protein glutathionylation ( protein - ssg ) and has been linked to numerous disease states associated with oxidative stress . as shown in figure 2(a ) , immunohistochemical staining for protein - ssg reveals a marked increase in zone 3 hepatocyte staining . although the impact of this modification is unknown , current theories propose protein - ssg to be a protective mechanism , shielding critical cysteine residues from more permanent oxidative modifications , that is , 4-hne . mechanisms behind protein - ssg remain unknown ; however , recent reports have proposed a role for the glutathione s - transferase ( gst ) and glutaredoxin ( grx ) family of enzymes [ 38 , 39 ] . we , therefore , measured these enzymes in our disease model . as shown in figures 2(b ) and 2(c ) , a significant increase in pan - gst and grx activities was observed in the ethanol - fed mice following 6 weeks , while no significant difference was observed at weeks 1 or 3 . the expression of these enzymes was also assessed via immunoblotting , revealing no significant differences in protein expression ( data not shown ) . these data demonstrate , for the first time , protein - ssg as a consequence of sustained ethanol ingestion and provide novel avenues for research in the field of ald . the effects of ethanol consumption and 4-hne on proteasome activity have been previously documented [ 40 , 41 ] . immunohistochemical staining presented in figure 1(c ) reveals a significant increase in 4-hne protein adducts , suggesting a mechanism for altered protein folding following sustained ethanol consumption . to investigate the impact of sustained oxidative stress on protein folding in this model , immunohistochemical staining was performed using antiubiquitin antibodies to target misfolded proteins targeted for degradation . as shown in figure 3(a ) , marked accumulation of ubiquitinated proteins was found throughout zones 2 and 3 in ethanol - consuming mice following 6 weeks . these data suggest an increase in protein misfolding and/or erred protein load as noted by an increasing need for protein disposal . given the large increase in protein ubiquitination at the week 6 time point , proteasome activity was quantified to determine if the effects on protein ubiquitination were due to altered disposal or increased protein misfolding . as shown in figure 3(b ) , no significant alterations in proteasome activity were found between the week 6 control and ethanol - fed mice . these data collectively suggest an increase in protein misfolding following ethanol ingestion , with large quantities of tagged protein being observed during pathologic situations . given these observations , it was our goal to investigate the role of the upr in our model for early - stage ald . upr signaling is not only limited to protein expression , as these pathways remain extremely complex and reliant on protein localization and posttranslational modifications . to assess these parameters , immunoblotting was conducted to evaluate upr protein expression , localization and phosphorylation status . as a positive control , a liver homogenate was utilized from a mouse , which received an intraperitoneal injection of tunicamycin ( tm ) . as shown in figure 4(a ) , the expression of the hallmark indicator for the upr , glucose regulated protein 78kda ( grp78 ) , showed no change in expression at any time point . the activation of ire1 , as measured by the relative expression level of its phosphorylated form ( p - ire1 ) , was also observed indicating no major role for this pathway following ethanol ingestion . to investigate the perk pathway , both total eif2 and activated eif2 ( p - eif2 ) were assessed . finally , nuclear localization of atf6 ( natf6 ) is the major indicator for the activation of this pathway following initiation of upr signaling . as shown in figure 4(a ) , no change in natf6 was observed between control and ethanol - fed mice , despite a potent activation of this protein following tm injection . the er stress response has been shown to lead to a host of cellular responses , including steatosis [ 43 , 44 ] . relevant to the pathologies associated with early - stage ald , lipogenic mediators initiated by upr signaling were investigated ( figure 4(a ) ) . no significant change in the nuclear localization of srebp1 ( nsrebp1 ) or nsrebp2 was observed between control and ethanol - fed mice at any time point . although a trending increase in srebp1 activation was observed , blot densitometry revealed no significant difference among treatment groups ( data not shown ) . previous reports have shown a clear association between the er stress response and oxidative stress . this link is thought to occur through the induction of the ero1 proteins ( ero1 and ero1 ) and nox4 . to investigate a possible separation between these responses , the expression of ero1 , ero1 and nox4 was assessed via immunoblotting . as shown in figure 4(b ) , no significant change in expression was observed , indicating that the observed oxidative stress associated with early - stage ald precedes the involvement of upr signaling . previous work has supported roles for oxidative stress and er stress in the etiology of ald [ 4 , 5 , 8 , 13 , 14 , 45 , 46 ] . these pathological processes are intrinsically linked , with each capable of activating the other . in order to elucidate the mechanisms involved in the initiation of ald hepatopathy , our aim was to focus on the earliest initiating stages of the disease where therapeutic intervention is typically most effective . this 6-week lieber - decarli model for early - state ald represents the earliest initiating stages of disease progression as shown through the documented sustained ingestion of substantial quantities of alcohol resulting in significant elevations in plasma alt , liver triglycerides and increased liver : body weight ratio . histological analyses revealed a marked increase in pan - lobular hepatic lipid accumulation consistent with the early signs of steatosis . collectively , these data demonstrate a reliable model for early - stage ald , which allowed for further investigation into the parameters associated with the initiating stages of disease progression . oxidative stress is a well - recognized outcome of chronic ethanol ingestion and is likely to play a major role in ethanol - mediated liver damage [ 4 , 5 , 8 ] . previous works utilizing n - acetylcysteine have demonstrated a protective role for antioxidant therapies in ethanol - mediated liver injury [ 47 , 48 ] . these studies revealed a significant decrease in alt measurements , steatosis , and tbars content . while these data do not definitively prove that oxidative stress is responsible for the early - initiating stages of disease development , a strong correlation can be established . data presented here reveals a significant decrease in gsh following 6 weeks of ethanol consumption , indicating a decrease in the overall antioxidant capacity of the liver at this time point . further , a significant decrease in the activity of cbs was observed at the week 6 time point , demonstrating a potential decrease in the gsh precursor cysteine . previous work conducted on cbs has shown a decrease in enzymatic activity following other hepatic insults such as a methionine - choline - deficient diet and other cellular stresses [ 49 , 50 ] . we postulate that the effects on cbs activity in the control animals may be due to the high fat content in the control diet . more importantly , the effects of this diet on cbs activity appear to be exacerbated following ethanol consumption . the availability of cysteine is often the rate - limiting determinant in gsh biosynthesis , and these effects on cbs activity offer a potential novel mechanism for the observed decrease in gsh following sustained ethanol ingestion . the decrease in gsh was consistent with an increase in lipid peroxidation , as demonstrated by increased staining of 4-hne modified proteins in the livers of ethanol - fed mice . previous reports by backos et al . have highlighted the effects of 4-hne on the activity of gcl utilizing cultured cells ; gcl activity was found to be significantly increased following treatment with 4-hne , despite a significant depletion of cellular gsh . our findings revealed a similar result , where gcl activity was found to be significantly increased despite no change in the overall expression of the enzyme . although the effects of 4-hne on gcl activity are noted , the precise role of aldehyde adduction in the alcoholic liver remains to be fully characterized . following 6 weeks of ethanol ingestion , hepatic pan - lobular steatosis is observed , with larger lipid droplets present throughout zones 1 and 2 . these macrosteatotic vesicles are likely contributing to the increased lipid peroxidation observed , as demonstrated by staining with 4-hne throughout these zones . it should be noted as well that cyp2e1 is found almost exclusively throughout zone 3 and is completely absent in zones 1 and 2 . this may , in part , provide a likely explanation for the observed increase in zone 3 staining of protein - ssg , whereby these modifications act to protect critical thiol residues . oxidative stress occurring in zone 3 has been documented by numerous other groups ; however , the staining of ubiquitinated proteins in this region remains a novel finding . it is conceivable that the increased oxidative stress in zone 3 is resulting in other damaging oxidative modifications stemming from cyp2e1-mediated free radical generation . at the current time , the precise mechanisms and rationale behind the increased ubiquitin staining in zone 3 are unknown . reports from our laboratory utilizing the lieber - decarli model in rats , however , have identified the er - resident protein disulfide isomerase ( pdi ) to be a target for modification by 4-hne in vivo ; this leads to a decrease in enzymatic activity and impaired protein folding . collectively , these data suggest a potential mechanism for oxidative - stress - induced upr signaling in rodent models for ald [ 51 , 52 ] . in the past decade , er stress has been associated with an increasing number of hepatic disease states , most notably ald [ 10 , 53 ] . previous research in mice by ji et al . has suggested a role for er stress in an intragastric model of ald [ 13 , 14 , 45 ] . however , the intragastric model represents a model for more severe pathologies of ald , as shown by a roughly 8-fold increase in serum alt ( versus an approximate doubling here ) and a significant increase in inflammation ( inflammation was not seen in our model ) . the transition from steatosis to steatohepatitis is considered to be the critical pathogenic determinant in ald furthering a need for therapeutic intervention during the initiating stages of disease progression . recent reports have suggested an association between the inflammatory response and upr signaling , outlining a mechanism similar to that with oxidative stress [ 5457 ] . consistent with early - stage ald , our data demonstrate clearly that the inflammatory response does not precede disease pathogenesis and upr induction in this model . to further understand the mechanisms behind ald progression , our research focused on the relative pathogenic contributions of er stress and oxidative stress during the initiating stages of the disease . previous work in rodent models of ald has hypothesized a role for the er stress response in the activation of lipogenic pathways . this was shown to occur through srebp1 activation and this mechanism was thought to be a major contributor to the observed hepatic steatosis [ 13 , 45 ] . to investigate these reports , immunoblotting for both hallmark and lipogenic upr signaling cascades revealed no significant activation during our time course model for ald despite the generation of significant pathologies , such as increased lipid accumulation and serum alt levels . taken together , these data validate a minimal role for the upr during the development of ethanol - induced steatosis in a murine model for early - stage ald . oxidative stress has gained considerable attention as a possible mechanism for the induction of various cellular responses , including the er stress response [ 8 , 1519 , 58 ] . additionally , upr signaling has been shown to lead to induction of the ero1 proteins and nox4 , furthering the oxidative stress burden in the cell . this oxidative protein folding relay has been estimated by tu and weissman to account for up to 25% of total cellular ros . this creates a vicious cycle of cellular derangements , where each response propagates the other . regarding the alcoholic liver , upr signaling and oxidative stress have been intimately associated with the progression of ald . to date , these responses have not been investigated in relation to the initiation of ald and therefore the relative role each has during early - stage ald is not understood . our data demonstrate a clear delineation between the er stress response and oxidative stress in early - stage ald , indicating that oxidative stress is a primary initiating factor responsible for the progression of ald . the observed increase in protein glutathionylation demonstrates a potential involvement for this posttranslational modification following increased oxidative stress and provides interesting avenues for research in this field . the effects of oxidative stress in our model were determined to be independent of upr induction , indicating that the er stress response may play a pivotal role during more advanced stages of disease progression .

alcoholic liver disease ( ald ) is a primary cause of morbidity and mortality in the united states and constitutes a significant socioeconomic burden . previous work has implicated oxidative stress and endoplasmic reticulum ( er ) stress in the etiology of ald ; however , the complex and interrelated nature of these cellular responses presently confounds our understanding of ethanol - induced hepatopathy . in this paper , we assessed the pathological contribution of oxidative stress and er stress in a time - course mouse model of early - stage ald . ethanol - treated mice exhibited significant hepatic panlobular steatosis and elevated plasma alt values compared to isocaloric controls . oxidative stress was observed in the ethanol - treated animals through a significant increase in hepatic tbars and immunohistochemical staining of 4-hne - modified proteins . hepatic glutathione ( gsh ) levels were significantly decreased as a consequence of decreased cbs activity , increased gsh utilization , and increased protein glutathionylation . at the same time , immunoblot analysis of the perk , ire1 , atf6 , and srebp pathways reveals no significant role for these upr pathways in the etiology of hepatic steatosis associated with early - stage ald . collectively , our results indicate a primary pathogenic role for oxidative stress in the early initiating stages of ald that precedes the involvement of the er stress response .

1. Introduction 2. Experimental Procedures 3. Results 4. Discussion

PMC3263621

the main goal of endodontics is to restore the function and esthetic of the involved tooth . from a biomechanical perspective , this means cleaning , shaping , and disinfection that would allow for three - dimensional obturation of the root canal system . one of the main reasons for the failure of root canal treatment is the inadequate removal of pulp tissue and microorganisms from the root canal system . it is therefore of utmost importance that the dentist has a thorough knowledge of root canal morphology of the tooth being treated . the mandibular first molar is a frequently treated tooth and has a wide variety of root canal configurations . variations in the morphology of the dental pulp are caused by genetic and environmental influences , and there is very definite need for clinicians to be made aware of the frequency of racially determined forms . the major variant in mandibular first molars is the presence of a supernumerary root that can be found distolingually and has a curve at the apex . this macrostructure , first mentioned by carabelli , is called radix entomolaris , which in general is smaller than distobuccal and mesial roots and can be separate from or partially fused with these other roots . the morphology and buccolingual width of the mesial root allow for intercanal communications and isthmuses . currently , the isthmus ( anastomosis ) is defined as a pulpal passageway connecting 2 or more canals in the same root . studies on the root canal anatomy of mandibular first molar have been performed on several populations . an extensive literature search showed that there is only one study on the root canal morphology of indian mandibular first molars . the root canal anatomy of indian teeth by clearing technique has not been studied to date except for studies on premolars and mandibular second molars . hence , the purpose of this study was to prepare detailed investigation of root canal anatomy of mandibular first molar in an indian population by using clearing technique and to compare these findings with the published reports of different population . one hundred and fifty mandibular first molars were collected from the department of oral and maxillofacial surgery of the government dental college and hospital , nagpur in india . it was ensured that the teeth belonged to indigenous indians , and no teeth from other minority ethnicities were included . the collection of every tooth was accompanied by a case record stating and confirming the ethnicity of the patients . the teeth were washed under tap water immediately after extraction and stored in 10% formalin ( qualigens fine chemicals , mumbai , india ) . the residual soft tissues , bone fragments , and calculus were cleansed and removed by curettes and ultrasonic scalers . the specimens were then placed in 5.25% sodium hypochlorite ( prime dental products pvt ltd . , mumbai , india ) for 24 hours in order to dissolve organic debris and pulp remnants . the specimens were first decalcified at room temperature in 5% nitric acid ( qualigens fine chemicals , mumbai , india ) that was changed daily for 3 - 4 days . the acid was agitated thrice daily with glass rod , and the end point of decalcification was determined by periodic radiographs . after completion of decalcification , the specimens were washed under running tap water for 4 hours to remove traces of nitric acid . the specimens were dehydrated using ascending concentrations of ethyl alcohol ( thermo fisher scientific india pvt ltd . , mumbai , india ) starting with 70% for 12 hours , followed by 90% for an hour and 3 rinses of 1 hour each for 100% . the dehydrated specimens then were placed in methyl salicylate ( rankem fine chemicals ltd . , new delhi , india ) which made them transparent after approximately 2 hours . mumbai , india ) was injected into the pulp chamber with a 27-gauge needle on disposable syringe . the dye then was drawn through the canal system by applying negative pressure to the apical end of the tooth with the help of vacuum suction system . the excess ink was removed from the root surface with gauze soaked in 100% ethyl alcohol . two separate canals leave the pulp chamber and join short of the apex to form one canal . one canal leaves the pulp chamber , divides into two within the root , and then merges to exit as one canal . type v. one canal leaves the pulp chamber and divides short of the apex into two separate and distinct canals with separate apical foramina . two separate canals leave the pulp chamber , merge in the body of the root , and redivide short of the apex to exit as two distinct canals . one canal leaves the pulp chamber , divides and then rejoins within the body of the root , and finally redivides into two distinct canals short of the apex . the specimens were vertically sectioned in buccolingual direction through the furcation , to separate the mesial and distal halves . sectioning was done to avoid the overlapping of mesial and distal or distolingual roots during photography . the results of this study are summarized in ( tables 1 , 2 and 3 ) . of the 150 mandibular first molars , 94.6% had two roots , and 5.3% had extra distal roots ( distolingual root or radix entomolaris ) . in addition , 64% of the cases had three root canals ( mesiobuccal , mesiolingual and distal ) , and 36% had four root canals ( mesiobuccal , mesiolingual , distobuccal and distolingual ) ( table 1 ) . both the mesial and distal roots showed wide variations in canal configuration . in mesial root , type iv configuration was most prevalent ( 54% ) followed by type ii ( 36.6% ) , type vi ( 8% ) , and type v ( 0.6% ) ( figure 1(a)1(d ) ) . type ( 2 - 1 - 2 - 1 ) ( figure 1(e ) ) . in distal root , type i configuration was most prevalent ( 65.3% ) followed by type ii ( 20.6% ) , type iv ( 9.3% ) , type v ( 3.3% ) , and type iii ( 1.3% ) ( figure 2(a)2(e ) ) . in the three rooted molars , all distolingual roots possessed type i ( 100% ) canal configuration ( table 2 ) . isthmi ( intercanal communications ) ( figure 1(f ) ) were found in 30% of the cases in mesial and 10% of the cases in distal roots . apical deltas ( figure 2(f ) ) were found in 10% of the cases in mesial and 6% of the cases in distal roots ( table 3 ) . the methods used to study root canal morphology are replication technique [ 3 , 9 ] , clearing technique [ 68 , 1015 ] , use of radiopaque dyes and radiographs [ 2 , 1620 ] , sectioning of teeth , and recently , spiral computed tomography ( sct ) and cone beam computed tomography ( cbct ) . additionally , it is not necessary to gain an access into the specimen with instruments , thus original form and relationship of the canals are maintained . these include development of opaque areas due to incomplete dehydration , which can be correctable by additional dehydration in 100% ethyl alcohol . another common problem is the development of opacity after air drying ; however , this is readily reversible by immersion in methyl salicylate solution . in the present study , the prevalence of three rooted mandibular first molars among the indian population was 5.3% , similar to the study of garg et al . . this figure is lower than several earlier studies [ 8 , 14 , 16 , 22 ] , but higher than that reported by skidmore and bjorndal for caucasians , zaatar et al . for kuwaitis , sperber and moreau for africans , and al - qudah and awawdeh for a jordanian population . in this study , it was found that 36% of mandibular first molars had four canals . these results are similar to those of hartwell and bellizzi , who reported 35.1% of teeth and had four canals . this value is lower than the findings of several earlier authors [ 14 , 15 , 20 , 22 ] , but higher than that reported by skidmore and bjorndal , zaatar et al . , sperber and moreau , gulabivala et al . , and reuben et al . owing to the high percentage of two distal canals , classical triangular access preparation during root canal treatment should be extended towards the distolingual direction in a rectangular form to improve canal identification . in mesial root , type iv configuration was most prevalent ( 54% ) followed by type ii ( 36.6% ) , type vi ( 8% ) , and type v ( 0.6% ) configuration . this is consistent with the findings of most of the earlier studies [ 3 , 7 , 8 , 1215 , 17 , 21 , 22 ] , except the studies by zaatar et al . and al - nazhan which reported type ii being the most prevalent followed by type iv . in the present study , one mesial root showed an additional configuration type ( 2 - 1 - 2 - 1 ) as described by gulabivala et al . . identification , preparation , and obturation of type iv , type ii , and type vi are relatively straightforward . however , identification of canals in type v , where the canal further divides within the root , is more difficult . the presence of gulabivala 's type ( 2 - 1 - 2 - 1 ) needs extra efforts , because failure to debride and disinfect this complex anatomy might have a direct effect on the treatment outcome . there are published reports indicating the presence of type viii configuration in the mesial root , with the incidence of 0.2% to 5% [ 7 , 8 , 1215 , 22 ] . but in the present study , none of the samples had three canals in the mesial root . the most prevalent configuration in the distal root was type i ( 65.3% ) followed by type ii ( 20.6% ) , type iv ( 9.3% ) , type v ( 3.3% ) , and type iii ( 1.3% ) configuration . in terms of type ii and type iv configuration , this figure is lower than the studies of caliskan et al . , ahmed et al . , and al - nazhan , but higher than that reported by skidmore and bjorndal , vertucci , gulabivala et al . , and pineda and kuttler . the external morphology of the distal root is more rounded than the mesial one and therefore less likely to accommodate two separate canals . however , type iii and type v configurations in distal root need extra efforts to negotiate and prepare . one of the two canals , the one most continuous with the large main passage , is usually amenable to adequate enlarging and filling procedure , the preparation and filling of the other canal is often extremely difficult . in the three rooted molars , all distolingual roots possessed type i ( 100% ) canal configuration . in the present study , isthmi ( anastomosis ) were observed in 30% of the cases in mesial , and 10% of the cases in distal roots . this figure is lower than several earlier studies [ 7 , 8 , 12 , 13 , 15 ] . apical deltas were observed in 10% of the cases in mesial and 6% of the cases in distal roots , which is similar to the findings of vertucci and caliskan et al . , but higher than that reported by pineda and kuttler , gulabivala et al . , and al - qudah and awawdeh . the presence of isthmi and apical deltas may be of clinical significance , because it may be difficult to debride and fill these ramifications adequately . the use of sodium hypochlorite , preferably agitated by ultrasonics may help to reach the uninstrumented parts of the root canal system . furthermore , these ramifications can be more satisfactorily obturated by using some thermoplasticized gutta - percha technique rather than cold lateral condensation of gutta - percha points . the root canal morphology of 150 indian mandibular first permanent molars shows higher incidence of four canals ( 36% ) and extradistal roots ( 5.3% ) . therefore , the clinician must always look for a second canal in the distal root of indian mandibular first molars . the most prevalent canal configuration in the mesial root was vertucci type iv ( 54% ) , and in distal root type i ( 65.3% ) . an additional configuration , gulabivala type ( 2 - 1 - 2 - 1 ) , was found as a rare entity ( 0.6% ) in mesial root . as isthmi and apical deltas were observed in high percentage in mesial and distal root , efficient delivery and activation of irrigants are more essential . variations in the number of roots or canals and teeth with unusual root canal configurations have a definite impact on treatment . to achieve long - term success , clinician must use all the armamentaria at their disposal to locate and treat the entire root canal system .

an in vitro study was performed to determine the number of roots , root canals per tooth , root canal configurations , and frequency of isthmi and apical deltas in mandibular first permanent molars in an indian population . hundred and fifty mandibular first permanent molars were collected and subjected to clearing technique . the cleared teeth were examined in a stereomicroscope under 7.5x magnifications . the canal configurations were categorized using vertucci 's classification . overall 94.6% of the mandibular first molars had two roots , and 5.3% had extradistal roots ( distolingual root ) . in addition , 64% of the specimens had three root canals , and 36% had four root canals . the most common canal configurations of mesial and distal roots were vertucci type iv ( 54% ) and type i ( 65.3% ) , respectively . clinician should be aware of the complex root canal morphology of mandibular first molars among the indian population before and during the root canal treatment .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

PMC2141774

the bssmc cdna was cloned from genomic dna by pcr , using pfu polymerase ( lu and erickson , 1997 ) and adding ndei and bamhi sites to allow subcloning into the expression vector pet11 ( studier et al . , 1990 ) . when induced in bl21 at 37c , the protein was abundantly expressed but totally insoluble , and attempts to solubilize and renature it were unsuccessful . however , when the bacteria were maintained at 22c during induction , 1/3 of the protein remained soluble . the bacterial supernatant was partially purified by passing over a sephacryl hr-500 ( pharmacia biotech , piscataway , nj ) column in 20 mm tris , 100 mm nacl , ph 8 , where the bssmc eluted ahead of most bacterial proteins . the bssmc from the leading and trailing sephacryl fractions sedimented identically at 6.3 s on glycerol gradients , indicating a homogeneity of the expressed molecules . the sephacryl fractions showed some contamination , and in two cases were further purified by chromatography on mono q ( pharmacia biotech ) , where it eluted at 0.20.25 m nacl . however , the best preparations for em were obtained by running the sephacryl - purified bssmc directly on glycerol gradients . mukbcoil is missing both the nh2- and cooh - terminal domains and includes only the coiled coils and hinge ( aa 3191125 ; our analysis of heptad repeats was slightly different from the coil prediction of dnastar [ madison , wi ] , fig . 1 ) . mukbcoil tended to aggregate by sticking to small bits of bacterial debris ; however , some largely monomeric fractions could be obtained from glycerol gradient sedimentation . ( we also made a slightly larger coil construct , keeping the nh2 terminus at 319 and extending the cooh terminus to aa 1256 , which includes the last predicted segment of coiled coil [ fig . 1 ] . this longer coil segment was highly aggregated into rosettes , and there were no monomers . we concluded from this that the extra segment of predicted coiled coil is probably missing a partner to pair with , and therefore denatured , leading to aggregation . ) we then used the shorter mukbcoil as the basis for a chimera , fn - mukbcoil , in which the 40-kd cell adhesion domain of fibronectin , fn7 - 10 ( leahy et al . , we purified mukb as previously described using the expression plasmid pax814 kindly provided by dr . sota hiraga , kumamoto university , kumamoto , japan ( niki et al . , 1992 ) . we substituted a mono q column for the deae sephacel and mukb eluted at 0.4 m nacl . the best em results were obtained with material from the mono q column , subsequently sedimented over glycerol gradients . mukbcoil and fn - mukbcoil were purified by sephacryl chromatography followed by glycerol gradient sedimentation , omitting the mono q step . sedimentation coefficients were estimated by zone sedimentation through 1540% glycerol gradients in 0.2 m ammonium bicarbonate ( using the model sw50.1 or sw55.1 rotor ; beckman instruments , fullerton , ca ) ( erickson and briscoe , 1995 ) . standard proteins catalase ( 11.3 s ) and bsa ( 4.6 s ) were run in the same gradient as the samples , and the s value was estimated by linear interpolation between these standards ( sedimentation of other proteins has shown our gradients to be linear in this region ) . the stokes ' radius , rs in nm , was estimated by gel filtration on a superose 6 column using the following standards : thyroglobulin , rs = 8.5 nm ; catalase , rs = 5.2 nm ; and aldolase , rs = 4.8 nm . s and rs were used to calculate an experimental molecular weight as described by siegel and monte ( 1966 ) ( see ohashi and erickson , 1997 , for an example and our assumed parameters ) . samples from the glycerol gradients were rotary shadowed ( fowler and erickson , 1979 ) and photographed at 50,000 magnification in an electron microscope ( model 301 ; philips electron optics , mahwah , nj ) . 2 shows the purification of bssmc after overexpression in e. coli using the pet system . because the protein is so large and elongated , the gel filtration chromatography results in a substantial purification from the smaller bacterial proteins . a final step of glycerol gradient sedimentation resulted in fractions that showed only small contamination with other protein bands by sds - page and little obvious contamination by em . the sedimentation coefficient of bssmc was 6.3 s , based on four determinations ranging from 6.2 to 6.4 s , and excluding three earlier results from 7 to 8 s. the sedimentation coefficient of mukb was 9.6 s , based on six measurements ranging from 9.2 to 10.3 s , and excluding two at 6.7 and 7.5 s. in several mukb preparations , some of the protein was aggregated into rosettes , which sedimented ahead of the monomers . the stokes ' radius was measured for bssmc , mukb , and fn - mukbcoils by chromatography on a calibrated gel filtration column . the molecular masses calculated from these experimental values of s and rs are close to those predicted for a homodimer of each protein ( table i ) . the hydrodynamic data for these full - length proteins and other constructs are collected in table i and addressed further in the discussion . the fraction of molecules in the different conformations varied considerably from one field to another and is probably affected by the local conditions as the molecules are deposited on the mica and dried . both mukb and bssmc show the same three or four characteristic conformations , which is strong evidence that their basic structure is the same . the first conformation , which we call folded - rod , is a rod - shaped molecule with a large globular domain at one end and a smaller one at the other ( fig . the second conformation , which we call coils - spread , has similar large and small globular domains , but the rod connecting them is now split into two thinner rods ( fig . a third conformation was seen frequently for bssmc but was rare for mukb ( fig . the rods are together over most of the molecule but veer apart near the pair of globular domains . the fourth conformation , which we call open - v , has both the rod and the large globular domain split into two and splayed apart ( fig . 3 f ) . 3 f shows the structure of the molecule most clearly and explains the other forms . the globular domain in the middle is identified as the hinge , and the thin rods extending on each side are each a coiled coil . at the end of the coiled coils are the globular domains , discussed below . the coils - spread conformation is now interpreted as having the globular domains attached to each other but the two coiled coils separated and bowed out . the folded - rod conformation , giving the simple rod shape , is produced when the globular domains are attached to each other and the two coiled coils lie close to each other . measurements of several aspects of the molecules are tabulated and explained in table ii and will be addressed in the discussion . we initially anticipated that the coiled coils would be parallel since all known long coiled coils are parallel . this arrangement would place the two nh2-terminal domains at one end and the two cooh - terminal domains at the other end of an open - v molecule . we therefore expected to see some difference in size or shape in the terminal globular domains that would indicate this polarity . we also expected to see some difference in the lengths of the two arms since they would be formed from different segments of coiled coils . instead , we were surprised to find that both bssmc and mukb showed a striking symmetry . the arms were indistinguishable in length , and most importantly the domains at the two ends always appeared identical ( fig . 3 f ) . some molecules showed a simple globular domain on each arm , but the best images of both bssmc and mukb showed division of the terminal domain into a larger terminal globe and a smaller one somewhat closer to the hinge . remarkably , whenever the two - part domain structure could be resolved on one arm , an identical structure was seen on the other ( fig . the apparent twofold symmetry would only be possible with an antiparallel arrangement of the coiled coils . to address the question of polarity , we prepared constructs of mukb with markers at the nh2 terminus . we had already prepared a truncated mukb , mukbcoil , in which we had deleted both the nh2- and cooh - terminal domains , leaving only the two coiled - coil segments and the hinge . this molecule frequently appeared as a rod where the hinge was obvious as a single globular domain ; sometimes the two coiled - coil segments splayed apart in an open - v or coils - spread conformation ( fig . 4 a ) . the mukbcoil does not indicate the polarity because the nh2 and cooh termini are both truncated helices . to visualize the polarity , we made a new chimeric protein , fn - mukbcoil , in which we fused fn7 - 10 onto the nh2 terminus of mukbcoil . fn7 - 10 , a 40-kd fragment of fibronectin , is well characterized by x - ray crystallography ( leahy et al . , 1996 ) as a rod - shaped molecule about 3 nm in diameter and 14 nm long , which should easily stand out from the thinner coiled coil . if the molecules were a parallel coiled coil , we expected to see two fat fn rods projecting from one end . if they were antiparallel , we expected to see one fat rod at each end . the fn - mukbcoil molecules visualized in the em were nearly all in the open - v conformation and showed the fn segment at each end ( fig . 4 b ) this provides the most compelling evidence for the antiparallel arrangement of the coiled coils . there were several interesting contrasts between the structures of mukbcoil and fn - mukbcoil . mukbcoil tended to aggregate , sedimenting as a smear from 8 to 15 s , and the heavier fractions appeared as rosettes with the terminal segments of the coil aggregated at the center and the hinge projecting out . we believe that the ends of these coils are sticky , probably because we misjudged the termini of the coils . the sticky ends were also evident in single molecules , since the two ends usually remained in contact giving the folded - rod or coils - spread conformations ( fig . , fn - mukbcoil sedimented as a sharp peak at 7 s , and the molecules were unaggregated and primarily in the open - v conformation in the em . thus , the fn segment at the terminus of the rods seems to block the sticky sites and prevent association either within or between molecules . in addition to demonstrating the antiparallel arrangement of the coiled coils , fn - mukbcoil provides definitive proof that the hinge is flexible . 5 shows a histogram of the angles of the two arms measured for different molecules . we do not believe the apparent peaks and dips are significant , and we conclude that the angles are probably randomly distributed . it is perhaps of interest to ask whether the hinge can open to more than 180 , but these measurements can not distinguish 270 from 90. we conclude that the hinge is quite flexible between 1 and 180 and perhaps can bend even further . the frequency of the different conformations seen by em varied from field to field , but in most cases the folded - rod was the predominant conformation . the open - v conformation was the most important for demonstrating the fundamental structure of the molecule . these open - v molecules also demonstrate the potential of the terminal domains to separate and undergo a scissoring motion , which is likely to be important for biochemical functions . the hydrodynamic data are consistent with the elongated shape seen by em and provide insight into the question of whether the molecule is in the folded - rod or open - v conformation in solution . the shape of a molecule is best indicated by the frictional ratio f / fmin , where fmin is the frictional coefficient of an unhydrated sphere of the same mass and density as the protein in question ( tanford , 1961 ) . f / fmin was calculated from the measured sedimentation coefficient , and the mass was predicted from the subunit aa sequence . values are presented in table i for the four molecules we purified and for the 8-s xcap - c/ xcap - e heterodimer ( hirano et al . , 1997 ) . the fn - mukbcoil provides an important benchmark for comparison because em indicated that this construct was entirely in the open - v conformation . we believe that its f / fmin = 2.3 is characteristic of the open - v conformation . the lower values of 1.92.0 for mukb and xcap are probably characteristic of the folded - rod conformation . the f / fmin = 2.5 for bssmc is the largest of the group and strongly suggests that this molecule is open - v in solution . also , both bssmc and fn - mukbcoil have a larger stokes ' radius than mukb , which is consistent with the interpretation that they are open - v , and mukb is folded - rod in solution . the only contradiction is that most of the bssmc molecules appeared to be folded - rods in the em . it is possible that the folded - rod conformation of bssmc was generated by the high glycerol and salt concentrations when the specimens were dried on the mica . there were two possibilities for the mechanics of the hinge : it could be quite flexible , permitting free scissoring motion of the two coiled coils , or it could be relatively rigid , locking the coils into the parallel configuration seen in the folded - rod . a flexible hinge was implied by the coils - spread conformation , which frequently showed an angle of 60 or more for the coils at the hinge . the flexibility of the hinge was demonstrated most convincingly by the fn - mukbcoils construct , which appears by em to be entirely in the open - v conformation . since the hinge is flexible , the folded - rod conformation must be stabilized by an interaction of the terminal domains with each other . the measurements of the molecules in table ii show that mukb is longer than bssmc ( 65 vs. 58 nm ) , which is consistent with its larger mass and longer estimated coiled coil ( fig . 1 ) . we particularly wanted to determine the length of the coiled coil , as a guide to aligning the sequences . as shown in table ii , the measurement of the thin rod from the hinge to the outer globular domain ( subtracting the diameters of these domains from the length of the whole molecule ) gives 51 and 41 nm for mukb and bssmc . these would actually estimate the minimum length of the coiled coil since the coils could extend into the hinge and globular domain . using the spacing of 0.15 nm per aa in an helix , these lengths would predict coiled coils of 340 aa for mukb and 273 aa for bssmc . this construct included aa 319 1125 , extending in both directions slightly beyond the entire 330334-aa coiled - coil segments indicated in fig . 1 . however , the length of the coil measured from this construct was only 41 nm , corresponding to 273 aa . it therefore seems likely that the short predicted coil from 1205 1243 , which is missing from mukbcoil , is a part of the 51-nm rod of the whole mukb . it is not clear what this segment would be pairing with to make a coiled coil . it is interesting that a mutation d1201n , just before this predicted coil segment , completely disrupts mukb function ( yamanaka et al . , 1994 ) . the precise alignment of the coiled coils is complicated by the appearance of gaps in the predicted coils . some of these gaps may actually form coils and continue the coiled coil , but others may bud out , shortening the length of the rod . most of these interruptions are small and might not be visible even if they did fold into a globular domain . we note that the thin rods of the open - v molecules appear as a uniform , thin diameter between the hinge and the two globular domains at the ends . the heterodimeric smcs of eukaryotes pose additional ambiguity in alignment of the coils since there are now four separate sequences that have to form the two coiled coils . as shown in fig . 1 , reasonable alignments can be made for cut3/cut14 of schizosaccharomyces pombe and for xcap - c / xcap - e of xenopus laevis . the following two sections diverge from the structural analysis to discuss general features of the smc family of proteins . from the databases , we have collected 18 eukaryotic smc sequences , 6 bacterial sequences , and 4 archaeal sequences that appear to be bona fide smcs : mukb from e. coli , which has the smc structure but limited sequence identity ; and 8 sequences that show limited sequence identity at the nh2- and cooh - terminal domains and have long coiled - coil segments between them . we did a separate sequence alignment of the nh2-terminal domain of 230 aa and the cooh - terminal domain of 80 aa from each of the 34 sequences . the sequences were aligned by the clustal algorithm of dnastar and then adjusted by hand . 6 shows the two independently derived phylogenetic trees from the nh2- and cooh - terminal sequence alignments ( trees were drawn with the drawtree algorithm of phylip , http://evolution.genetics.washington.edu/phylip.html ) . the trees from the nh2- and cooh - terminal domains are remarkably similar , and a tree calculated from the hinge region ( not shown ) also shows the same grouping . all three trees cluster the smc sequences into six groups of smc proteins and a group of outliers . the six - smc groups comprise four eukaryotic smc groups , each containing one of the four s. cerevisiae smcs ; a group of bacterial smcs ; and a group of mostly archaeal smcs ( which , however , also includes the cyanobacterium synechocystis ) . we have labeled the eukaryotic groups 14 , corresponding to the s. cerevisiae smc in the group . ( this is consistent with the previously proposed nomenclature of koshland and strunnikov and a recent review by jessberger et al . ) b is for the bacterial group , and a is for the mostly archaeal group . ( 1998 ) have proposed that there are two classes of heterodimers : smc2/4 and smc1/3 . there are three well - established examples of pairing a member of the smc2 group with a member of the smc4 group : xcap - e + xcap - c ( hirano and mitchison , 1994 ) , cut14 + cut3 ( saka et al . , 1994 ) , and mix-1 + dpy-27 ( lieb et al . , 1998 ) . it is attractive to speculate that an smc of group 2 always pairs with a group 4 partner . mix-1 provides an interesting variation , since it is known to pair with dpy-27 for its role in dosage compensation , but it probably has a different smc partner for its role in mitosis ( lieb et al . , 1998 ) . c. elegans has a second smc in group 4 , so far identified only from genomic sequencing . heterodimers of a group 3 and group 1 smc have been identified for bovine smc ( jessberger et al . , 1998 ) and xenopus ( losada et al . , 1998 ) . in s. cerevisiae , smc3 and smc1 were each identified in a screen for mutants affecting sister chromatid cohesion ( michaelis et al . , 1997 ) , consistent with their forming a pair . a possible exception to the generalization is that smc2 from s. cerevisiae has been shown to associate both with itself and with smc1 ( strunnikov et al . , 1995 ) . it was not determined that either of these was a specifically heterodimer association , but this observation raises the possibility that heterodimer associations may be more complex than simply two classes . of 18 bacterial or archaeal genomes completely or almost completely sequenced , 13 have a clear smc , and 3 have no smc but a mukb ( which we now consider a divergent smc ; see also britton et al . , 1998 ) . methanococcus thermoautotrophican has only a distantly related protein but no smc or mukb , and borelia burgdorferi has an smc - like protein that is shorter than most and branches ambiguously between bacterial smcs and outliers . helicobacter pylori has nothing matching the conserved nh2 or cooh termini of smc , mukb , or the distantly related proteins ( erickson , h.p . , unpublished sequence analysis ; sequence data for partially completed genomes were obtained from the institute for genomic research website at http://www.tigr.org ) . the most distinctive structural feature of the smc proteins is the presence of five domains : nh2-terminal , long coiled coil , hinge , long coiled coil , and cooh - terminal . the coiled - coil segments of smc proteins are not highly conserved ; however , the nh2-terminal , cooh - terminal , and hinge domains show a high level sequence match over several motifs that seem characteristic of the smc proteins . for the present discussion , we will not show the full alignment but will designate the motifs according to aa numbers of the bssmc sequence ( swiss prot p51834 ) . in the nh2-terminal domain , the most striking motifs are fks ( 1113 ) , gxngsgksn ( 3139 ) , which is the walker a motif for ntp binding , and the dipeptide qg ( 143144 ) . the fks and gxngsgksn are almost totally conserved in the bona fide smc proteins , and even the outliers show mostly conservative substitutions . a most intriguing sequence motif , not previously noted , is the qg , which is conserved in all 26 smc sequences examined . it is also conserved in all three known mukb proteins ( from e. coli , h. influenzae , and vibrio cholerae [ sequence data for v. cholerae were obtained from the institute for genomic research website at http://www . qg is conserved in methanococcus jannaschii 1322 and the sulfolobus protein , but not in methanobacter thermoautotrophican nor in any of the rad proteins . we suggest that this qg is a diagnostic signature of the smc proteins and is another argument for including mukb in this group . the hinge region shows no extended conserved motif , but the segment from aa 517 to 666 contains several isolated aa that are highly conserved . it is interesting that rad18 and rhc18 show a hinge region that matches that of the smc proteins . mukb has a 125-aa noncoil segment that obviously functions as a hinge , but it shows no sequence similarity to the smc hinge sequence . the cooh - terminal domain as indicated in fig . 1 includes 200300 aa from the end of the coiled coil , but only a short terminal segment of about 80 aa shows significant sequence identity . this segment begins with the highly conserved lsgg ( 10911094 ) and then the pxphhhhdehdaald ( 11121126 ) , where h is a hydrophobic aa . the hhhhd may correspond to the walker b site , which is very loosely defined as an aspartic acid preceded by four hydrophobic residues . ( 1995 ) for a more detailed analysis of this motif and its use in placing the smc proteins in a larger family of transporters , helicases , and repair enzymes . a recent study of the e. coli dna - processing protein sbcc has proposed including it as a member of the smc family ( connelly et al . , 1998 ) . this protein falls among the outliers in our alignments of nh2- and cooh - terminal domains ( fig . 6 ) , and moreover has no functional hinge ( see below ) . although one could expand the definition of the smc family to include the rad proteins and other outliers , we suggest for the present to limit the smc designation to the family members defined by the above characteristics , including specifically a functional hinge . the antiparallel orientation seems preferred for the short , interacting helices found in globular proteins , but coiled - coil proteins are predominantly parallel . two exceptions are the antiparallel , 35-aa coiled coil projecting as a helical arm of the bacterial seryl trna synthetases ( oakley and kim , 1997 ) and a 45-aa coiled coil of f1-atpase ( abrahams , 1994 ) . the 300-aa coiled coils of the smc proteins demonstrate for the first time that the antiparallel arrangement can be used to form very long coiled coils . we predict that the eukaryotic smc heterodimers will have the same antiparallel coiled coil structure ( fig . 1 ) , and this may also be true for the several proteins ( rad50 , rad18 , e. coli sbcc , m. jannaschii 1322 ) indicated as outliers in the phylogenetic tree . a recent study of one of these outliers , sbccd , included an electron micrograph that provides some important insights ( connelly et al . , 1998 ) . the sbccd showed two large globular domains separated by a single coiled coil 80 nm long . although the sequence indicates a short break in the middle of this coiled coil , the images show no indication of a hinge in the structure . the coiled coil shows a gentle curvature that can bring the terminal domains within 40 nm of each other , but this molecule seems incapable of achieving a folded - rod conformation . the question of parallel or antiparallel arrangement was not addressed in that study , and indeed the structure is described as however , there is no evidence of asymmetry in the structure ; the globular domains at the two ends actually appear to be the same size , consistent with an antiparallel arrangement of the coiled coils in sbccd . it is also interesting to note that the antiparallel structure provides a mechanism for bringing the proposed walker a and b motifs together because the nh2- and cooh - terminal domains are paired at each end of the molecule . alternatively , in the smc proteins they could be brought together in the folded - rod conformation . however , since sbccd seems incapable of forming the folded - rod conformation , the antiparallel structure seems the only possibility for bringing its nh2- and cooh - terminal domains together . we should caution that the resolution of the analysis is not sufficient to actually demonstrate contact of the domains , much less a functional association of the walker a and b motifs . the antiparallel coiled coil arrangement also suggests the possibility of a twofold axis of symmetry passing through the hinge . the twofold symmetry could be exact for the homodimers and could be approximate for the heterodimers . this would relate the nh2- and cooh - terminal domains on opposite arms by a 180 rotation , bringing identical faces into contact in the folded - rod conformation . perhaps the most important feature of the new model is that the molecule is not a polar structure , with an atp - binding domain at one end and a dna - binding domain at the other , but each terminus of the molecule contains a complete and identical functional unit . this means that the two ends of the molecule can operate identically on two strands of dna , separated by 100 nm for the fully open molecule , or brought into contact in the folded - rod . one function well established for 13-s xcap condensin is the generation of dna supercoils in vitro ( kimura and hirano , 1997 ) , and this may be the basis for dna condensation . the symmetrical molecule with two complete functional units should suggest novel mechanisms for how the smc complex might operate on dna to generate supercoils , and for how related complexes might manipulate dna for cohesion or repair . the frequency of the different conformations seen by em varied from field to field , but in most cases the folded - rod was the predominant conformation . the open - v conformation was the most important for demonstrating the fundamental structure of the molecule . these open - v molecules also demonstrate the potential of the terminal domains to separate and undergo a scissoring motion , which is likely to be important for biochemical functions . the hydrodynamic data are consistent with the elongated shape seen by em and provide insight into the question of whether the molecule is in the folded - rod or open - v conformation in solution . the shape of a molecule is best indicated by the frictional ratio f / fmin , where fmin is the frictional coefficient of an unhydrated sphere of the same mass and density as the protein in question ( tanford , 1961 ) . f / fmin was calculated from the measured sedimentation coefficient , and the mass was predicted from the subunit aa sequence . values are presented in table i for the four molecules we purified and for the 8-s xcap - c/ xcap - e heterodimer ( hirano et al . , 1997 ) . the fn - mukbcoil provides an important benchmark for comparison because em indicated that this construct was entirely in the open - v conformation . we believe that its f / fmin = 2.3 is characteristic of the open - v conformation . the lower values of 1.92.0 for mukb and xcap are probably characteristic of the folded - rod conformation . the f / fmin = 2.5 for bssmc is the largest of the group and strongly suggests that this molecule is open - v in solution . also , both bssmc and fn - mukbcoil have a larger stokes ' radius than mukb , which is consistent with the interpretation that they are open - v , and mukb is folded - rod in solution . the only contradiction is that most of the bssmc molecules appeared to be folded - rods in the em . it is possible that the folded - rod conformation of bssmc was generated by the high glycerol and salt concentrations when the specimens were dried on the mica . there were two possibilities for the mechanics of the hinge : it could be quite flexible , permitting free scissoring motion of the two coiled coils , or it could be relatively rigid , locking the coils into the parallel configuration seen in the folded - rod . a flexible hinge was implied by the coils - spread conformation , which frequently showed an angle of 60 or more for the coils at the hinge . the flexibility of the hinge was demonstrated most convincingly by the fn - mukbcoils construct , which appears by em to be entirely in the open - v conformation . since the hinge is flexible , the folded - rod conformation must be stabilized by an interaction of the terminal domains with each other . the measurements of the molecules in table ii show that mukb is longer than bssmc ( 65 vs. 58 nm ) , which is consistent with its larger mass and longer estimated coiled coil ( fig . 1 ) . we particularly wanted to determine the length of the coiled coil , as a guide to aligning the sequences . as shown in table ii , the measurement of the thin rod from the hinge to the outer globular domain ( subtracting the diameters of these domains from the length of the whole molecule ) gives 51 and 41 nm for mukb and bssmc . these would actually estimate the minimum length of the coiled coil since the coils could extend into the hinge and globular domain . using the spacing of 0.15 nm per aa in an helix , these lengths would predict coiled coils of 340 aa for mukb and 273 aa for bssmc . this construct included aa 319 1125 , extending in both directions slightly beyond the entire 330334-aa coiled - coil segments indicated in fig . 1 . however , the length of the coil measured from this construct was only 41 nm , corresponding to 273 aa . it therefore seems likely that the short predicted coil from 1205 1243 , which is missing from mukbcoil , is a part of the 51-nm rod of the whole mukb . it is not clear what this segment would be pairing with to make a coiled coil . it is interesting that a mutation d1201n , just before this predicted coil segment , completely disrupts mukb function ( yamanaka et al . , 1994 ) . the precise alignment of the coiled coils is complicated by the appearance of gaps in the predicted coils . some of these gaps may actually form coils and continue the coiled coil , but others may bud out , shortening the length of the rod . most of these interruptions are small and might not be visible even if they did fold into a globular domain . we note that the thin rods of the open - v molecules appear as a uniform , thin diameter between the hinge and the two globular domains at the ends . the heterodimeric smcs of eukaryotes pose additional ambiguity in alignment of the coils since there are now four separate sequences that have to form the two coiled coils . as shown in fig . 1 , reasonable alignments can be made for cut3/cut14 of schizosaccharomyces pombe and for xcap - c / xcap - e of xenopus laevis . the following two sections diverge from the structural analysis to discuss general features of the smc family of proteins . from the databases , we have collected 18 eukaryotic smc sequences , 6 bacterial sequences , and 4 archaeal sequences that appear to be bona fide smcs : mukb from e. coli , which has the smc structure but limited sequence identity ; and 8 sequences that show limited sequence identity at the nh2- and cooh - terminal domains and have long coiled - coil segments between them . we did a separate sequence alignment of the nh2-terminal domain of 230 aa and the cooh - terminal domain of 80 aa from each of the 34 sequences . the sequences were aligned by the clustal algorithm of dnastar and then adjusted by hand . 6 shows the two independently derived phylogenetic trees from the nh2- and cooh - terminal sequence alignments ( trees were drawn with the drawtree algorithm of phylip , http://evolution.genetics.washington.edu/phylip.html ) . the trees from the nh2- and cooh - terminal domains are remarkably similar , and a tree calculated from the hinge region ( not shown ) also shows the same grouping . all three trees cluster the smc sequences into six groups of smc proteins and a group of outliers . the six - smc groups comprise four eukaryotic smc groups , each containing one of the four s. cerevisiae smcs ; a group of bacterial smcs ; and a group of mostly archaeal smcs ( which , however , also includes the cyanobacterium synechocystis ) . we have labeled the eukaryotic groups 14 , corresponding to the s. cerevisiae smc in the group . ( this is consistent with the previously proposed nomenclature of koshland and strunnikov and a recent review by jessberger et al . , which did not include the bacterial and archaeal smcs . ) b is for the bacterial group , and a is for the mostly archaeal group . ( 1998 ) have proposed that there are two classes of heterodimers : smc2/4 and smc1/3 . there are three well - established examples of pairing a member of the smc2 group with a member of the smc4 group : xcap - e + xcap - c ( hirano and mitchison , 1994 ) , cut14 + cut3 ( saka et al . , 1994 ) , and mix-1 + dpy-27 ( lieb et al . , 1998 ) . it is attractive to speculate that an smc of group 2 always pairs with a group 4 partner . mix-1 provides an interesting variation , since it is known to pair with dpy-27 for its role in dosage compensation , but it probably has a different smc partner for its role in mitosis ( lieb et al . c. elegans has a second smc in group 4 , so far identified only from genomic sequencing . heterodimers of a group 3 and group 1 smc have been identified for bovine smc ( jessberger et al . , 1998 ) and xenopus ( losada et al . , 1998 ) . in s. cerevisiae , smc3 and smc1 were each identified in a screen for mutants affecting sister chromatid cohesion ( michaelis et al . , 1997 ) , consistent with their forming a pair . a possible exception to the generalization is that smc2 from s. cerevisiae has been shown to associate both with itself and with smc1 ( strunnikov et al . , 1995 ) . it was not determined that either of these was a specifically heterodimer association , but this observation raises the possibility that heterodimer associations may be more complex than simply two classes . of 18 bacterial or archaeal genomes completely or almost completely sequenced , 13 have a clear smc , and 3 have no smc but a mukb ( which we now consider a divergent smc ; see also britton et al . methanococcus thermoautotrophican has only a distantly related protein but no smc or mukb , and borelia burgdorferi has an smc - like protein that is shorter than most and branches ambiguously between bacterial smcs and outliers . helicobacter pylori has nothing matching the conserved nh2 or cooh termini of smc , mukb , or the distantly related proteins ( erickson , h.p . , unpublished sequence analysis ; sequence data for partially completed genomes were obtained from the institute for genomic research website at http://www.tigr.org ) . the most distinctive structural feature of the smc proteins is the presence of five domains : nh2-terminal , long coiled coil , hinge , long coiled coil , and cooh - terminal . the coiled - coil segments of smc proteins are not highly conserved ; however , the nh2-terminal , cooh - terminal , and hinge domains show a high level sequence match over several motifs that seem characteristic of the smc proteins . for the present discussion , we will not show the full alignment but will designate the motifs according to aa numbers of the bssmc sequence ( swiss prot p51834 ) . in the nh2-terminal domain , the most striking motifs are fks ( 1113 ) , gxngsgksn ( 3139 ) , which is the walker a motif for ntp binding , and the dipeptide qg ( 143144 ) . the fks and gxngsgksn are almost totally conserved in the bona fide smc proteins , and even the outliers show mostly conservative substitutions . a most intriguing sequence motif , not previously noted , is the qg , which is conserved in all 26 smc sequences examined . it is also conserved in all three known mukb proteins ( from e. coli , h. influenzae , and vibrio cholerae [ sequence data for v. cholerae were obtained from the institute for genomic research website at http://www . qg is conserved in methanococcus jannaschii 1322 and the sulfolobus protein , but not in methanobacter thermoautotrophican nor in any of the rad proteins . we suggest that this qg is a diagnostic signature of the smc proteins and is another argument for including mukb in this group . the hinge region shows no extended conserved motif , but the segment from aa 517 to 666 contains several isolated aa that are highly conserved . it is interesting that rad18 and rhc18 show a hinge region that matches that of the smc proteins . mukb has a 125-aa noncoil segment that obviously functions as a hinge , but it shows no sequence similarity to the smc hinge sequence . the cooh - terminal domain as indicated in fig . 1 includes 200300 aa from the end of the coiled coil , but only a short terminal segment of about 80 aa shows significant sequence identity . this segment begins with the highly conserved lsgg ( 10911094 ) and then the pxphhhhdehdaald ( 11121126 ) , where h is a hydrophobic aa . the hhhhd may correspond to the walker b site , which is very loosely defined as an aspartic acid preceded by four hydrophobic residues . ( 1995 ) for a more detailed analysis of this motif and its use in placing the smc proteins in a larger family of transporters , helicases , and repair enzymes . a recent study of the e. coli dna - processing protein sbcc has proposed including it as a member of the smc family ( connelly et al . , 1998 ) . this protein falls among the outliers in our alignments of nh2- and cooh - terminal domains ( fig . 6 ) , and moreover has no functional hinge ( see below ) . although one could expand the definition of the smc family to include the rad proteins and other outliers , we suggest for the present to limit the smc designation to the family members defined by the above characteristics , including specifically a functional hinge . the antiparallel orientation seems preferred for the short , interacting helices found in globular proteins , but coiled - coil proteins are predominantly parallel . two exceptions are the antiparallel , 35-aa coiled coil projecting as a helical arm of the bacterial seryl trna synthetases ( oakley and kim , 1997 ) and a 45-aa coiled coil of f1-atpase ( abrahams , 1994 ) . the 300-aa coiled coils of the smc proteins demonstrate for the first time that the antiparallel arrangement can be used to form very long coiled coils . we predict that the eukaryotic smc heterodimers will have the same antiparallel coiled coil structure ( fig . 1 ) , and this may also be true for the several proteins ( rad50 , rad18 , e. coli sbcc , m. jannaschii 1322 ) indicated as outliers in the phylogenetic tree . a recent study of one of these outliers , sbccd , included an electron micrograph that provides some important insights ( connelly et al . , 1998 ) . the sbccd showed two large globular domains separated by a single coiled coil 80 nm long . although the sequence indicates a short break in the middle of this coiled coil , the images show no indication of a hinge in the structure . the coiled coil shows a gentle curvature that can bring the terminal domains within 40 nm of each other , but this molecule seems incapable of achieving a folded - rod conformation . the question of parallel or antiparallel arrangement was not addressed in that study , and indeed the structure is described as however , there is no evidence of asymmetry in the structure ; the globular domains at the two ends actually appear to be the same size , consistent with an antiparallel arrangement of the coiled coils in sbccd . it is also interesting to note that the antiparallel structure provides a mechanism for bringing the proposed walker a and b motifs together because the nh2- and cooh - terminal domains are paired at each end of the molecule . alternatively , in the smc proteins they could be brought together in the folded - rod conformation . however , since sbccd seems incapable of forming the folded - rod conformation , the antiparallel structure seems the only possibility for bringing its nh2- and cooh - terminal domains together . we should caution that the resolution of the analysis is not sufficient to actually demonstrate contact of the domains , much less a functional association of the walker a and b motifs . the antiparallel coiled coil arrangement also suggests the possibility of a twofold axis of symmetry passing through the hinge . the twofold symmetry could be exact for the homodimers and could be approximate for the heterodimers . this would relate the nh2- and cooh - terminal domains on opposite arms by a 180 rotation , bringing identical faces into contact in the folded - rod conformation . perhaps the most important feature of the new model is that the molecule is not a polar structure , with an atp - binding domain at one end and a dna - binding domain at the other , but each terminus of the molecule contains a complete and identical functional unit . this means that the two ends of the molecule can operate identically on two strands of dna , separated by 100 nm for the fully open molecule , or brought into contact in the folded - rod . one function well established for 13-s xcap condensin is the generation of dna supercoils in vitro ( kimura and hirano , 1997 ) , and this may be the basis for dna condensation . the symmetrical molecule with two complete functional units should suggest novel mechanisms for how the smc complex might operate on dna to generate supercoils , and for how related complexes might manipulate dna for cohesion or repair . coiled - coil segments predicted by the program protean ( dnastar ) are shown as black rectangles . numbers above the vertical lines indicate the aa number , and numbers in parentheses between arrows indicates the total number of aa between the lines . the 275300-aa segment for bssmc and the 330335-aa segment of ecmukb were initially selected as the coiled coil because they matched the 41- and 51-nm lengths measured by em . however , measurements of the truncated construct mukbcoil ( see discussion ) indicate that the coiled coil of mukb probably includes the short segment 12051243 . the boundaries and alignment of the coiled - coil segments are therefore still ambiguous . heterodimeric smcs may also pair by the antiparallel coiled - coil arrangement , as illustrated for cut3/cut14 and xcap - e / xcap - c . about one - third of the bssmc is in the supernatant in these expressing bacteria grown at 22c . the next seven lanes show fractions from the sephacryl column , and the final three lanes show fractions from the glycerol gradient ( of the peak sephacryl fraction ) . m kd aa seq is the mass calculated for the presumed dimer from the aa sequence . m experim was calculated from these values ( siegel and monte , 1966 ) . f / fmin is the ratio of the experimental frictional coefficient ( determined from s ) to that of an unhydrated sphere of the same mass . selected fields are presented in a and b. ( c ) the most common conformation , folded - rod . ( d ) the coils - spread conformation . ( e ) molecules with the coils together but with the terminal domains split , which was seen reproducibly for bssmc . ( f ) the most informative open - v conformation . note the symmetry of the molecules in the open - v conformation : whenever one arm shows two small globular domains , the other arm does also . measurements of mukb and bssmc lengths and calculations all dimensions listed here have been corrected for the presumed 1-nm shell of metal . 1 . the most definitive measurement was the length of the whole molecule in the folded - rod conformation and the lengths of single arms in the open - v ; these were the same , and the measurements given here include both forms . 2 . the smaller globular domain was identified as the hinge and had the same diameter in all three conformations . 3 . molecules in the open - v conformation usually showed the terminal domain resolved into two globular domains . the length of the coiled - coil was estimated by subtracting the diameters of the hinge and outer globular domains from the length of the whole molecule . the length of the mukbcoil was measured from the outside of the globular hinge domain to the termination of the thin coil . this termination was sometimes ambiguous , hence the larger standard deviation of these measurements . the length of the coil was estimated by subtracting the diameter of the hinge ( 2 ) from the length of mukbcoil ( 5 ) . the larger ( nonhinge ) globular domain was approximately spherical in all conformations of bssmc , and its diameter is measure here . the coil of bssmc was estimated by subtracting the diameters of the hinge and outer globular domain from the length of the whole molecule . ( a ) mukbcoil , in which both the nh2- and cooh - terminal domains were deleted . the ends seem to be sticky and are frequently together ( right - hand images ) , although some open - v molecules could be found . ( b ) fn - mukbcoil , in which a thick segment of fibronectin was attached to the nh2 terminus of the coiled coil . this segment completely eliminated the stickiness of the ends , and all molecules are in the open - v configuration . the thick fn segment is seen projecting from each end , confirming the antiparallel coiled - coil arrangement . molecules of fn - mukbcoil in the open - v conformation were measured for the angle of the two arms . note that angles larger than 180 are possible but could not be distinguished , so they would be grouped with the smaller angle . separate trees are shown for the 220240-aa nh2-terminal domain ( flkrl ... lehve in bssmc ) , and the 80-aa cooh - terminal domain ( lsgge ... yssdt in bssmc ) . the circles indicate the six groups of smcs : 14 , eukaryal smcs ; b , bacterial ; a , archaeal . the group under the circle out are outliers , distantly related to smcs . each of the outlier sequences has an nh2-terminal domain with a related atp - binding motif , some limited sequence identity in the cooh - terminal domain , and long coiled coils separating the two , but usually no hinge . group 1 smc : smc1 ( s. cer ) , p41003 ; h. sapiens , s78271 ; xsmc1 ( x. la ) , af051784 . group 2 smc : smc2 ( s. cer ) , p38989 ; cut14 ( s. pom ) , p41003 ; mix-1 ( c. el ) , u96387 ; xcap - e ( x. la ) , p50533 ; scii ( chick ) , q90988 . group 3 smc : smc3 ( s. cer ) , p47037 ; smc3 ( s. pom ) , al009197 ; a.(e . ) nidulans , s65799 ; d. melanog . , u30492 ; bamacan ( rat ) , u82626 ; xsmc3 ( x. la ) , af051785 . group 4 smc : smc4 ( s. cer ) , u53880 ; cut3 ( s. pom ) , p41004 ; dpy-27 ( c. el ) , p48996 ; smc4a ( c. el ) , z46242 ; xcap - c ( x. la ) , p50532 . group b bacterial smcs : bacillus subtilis , p51834 ; mycobacterium tuberculosis , q10970 ; treponema palidum , orf00437 ; mycoplasma hyorhinis , p41508 ; mycoplasma pneumoniae , p75361 . group a ( mostly ) archaeal smcs : methanococcus jannaschii , u67604 ; aquifex aeolicus , ae000699 ; archeoglobus fulgidus , ae000995 ; synechocystis , d90905 ; pyrococcus horikoshii , d90905 . outliers proteins distantly related to smc : mukb ( e. coli ) , p22523 ; m. jannaschii 1322 , a64465 ; methanobacterium thermoautotrophican , ae000837 ; sulfolobus acidophilum , y10687 ; rad50 ( mouse ) , u66887 ; rad50 ( s. cer ) , p12753 ; rad18 ( s. pom ) , p53692 ; rhc18 ( s. cer ) , q12749 ; sbcc ( e. coli ) , p13458 . the model of the smc protein structure . the nh2- and cooh - terminal domains are shown schematically , without attempting to identify them with the two globular domains seen by em in some open molecules . the coiled - coil rods are rather rigid , but the hinge is quite flexible , permitting a scissoring movement with the coils separated at angles from 0 to 180 or more . the terminal domains associate with each other to lock the molecule into the folded - rod conformation , but this association is reversible , permitting transition to the open - v conformation .

structural maintenance of chromosomes ( smc ) proteins function in chromosome condensation and several other aspects of dna processing . they are large proteins characterized by an nh2-terminal nucleotide triphosphate ( ntp)-binding domain , two long segments of coiled coil separated by a hinge , and a cooh - terminal domain . here , we have visualized by em the smc protein from bacillus subtilis ( bssmc ) and mukb from escherichia coli , which we argue is a divergent smc protein . both bssmc and mukb show two thin rods with globular domains at the ends emerging from the hinge . the hinge appears to be quite flexible : the arms can open up to 180 , separating the terminal domains by 100 nm , or close to near 0 , bringing the terminal globular domains together.a surprising observation is that the 300amino acid long coiled coils are in an antiparallel arrangement . known coiled coils are almost all parallel , and the longest antiparallel coiled coils known previously are 3545 amino acids long . this antiparallel arrangement produces a symmetrical molecule with both an nh2- and a cooh - terminal domain at each end . the smc molecule therefore has two complete and identical functional domains at the ends of the long arms . the bifunctional symmetry and a possible scissoring action at the hinge should provide unique biomechanical properties to the smc proteins .

Materials and Methods Results Discussion Conformations and Dimensions Phylogenetic Tree of the SMC Family and Consideration of Heterodimers Conserved Sequences and the Defining Characteristics of SMC Proteins The Uniqueness of the Long, Antiparallel Coiled Coil Figures and Tables

PMC5361019

in sports like basketball and soccer , fatigue inevitably occurs during a game or practice session . certain lower extremity injuries , such as non - contact anterior cruciate ligament ( acl ) injuries , have a higher risk of occurrence in the final phase of games , e.g. , between 76 and 90 min of a soccer game1,2,3 . altered movement patterns caused by fatigue may explain non - contact acl injuries , as well as other injuries that occur during sporting activities . in contrast , to explain its effect on the lower extremities , evidence indicates that fatigue induces malalignment of the lower extremities and knee joints stability during sporting activities7,8,9 . furthermore , while athletes run in fatigue conditions , fatigue causes an increase in impact acceleration , which is regarded as the rapid deceleration of the tibia , during landings10 . moreover , lower extremity angles and the ground reaction force during landings are affected by fatigue11 , 12 . these findings indicate that fatigue alters lower extremity landing strategies and the ability to attenuate impact during landing . thus , the influence of fatigue during landing , an important movement , requires appropriate evaluation to consider measures to prevent lower extremity injuries . therefore , most clinicians use video analysis to evaluate lower extremity angles and alignments at specific times13,14,15 . however , lower extremity injuries may cause changes to angles and velocities over time , such as the direction change phase in sidestepping movements or the landing phase of a jump16 , 17 . it is also known that knee joint angular velocity variation during the landing phase is higher in females than in males18,19,20 and some studies have measured knee angular velocity to evaluate capacity to attenuate impact , which may be associated with knee injury risk6 , 18 , 19 . accordingly , lower extremity angular velocity during landing reflects the ability to attenuate impact because it results from rapid angular variations in the lower extremities during landing . therefore , lower extremity angles and angular velocities should be evaluated to identify biomechanical factors associated with lower extremity injuries . evidence indicates that fatigue causes changes in lower extremity kinematics , including angles and alignments during landing4 , 7 , 8 . fatigue may also alter lower extremity angular velocities and the ability to attenuate impact during landing . in our previous study , it has reported that fatigue might decrease the ability to attenuate impact by increasing the peak angular velocity in the direction of knee flexion during a single - leg jump landing21 . however , no studies to date have addressed the angular velocity variables of lower extremities , including hip and ankle joints , during fatigue conditions . thus , the purpose of this study was to evaluate the influence of fatigue on the lower extremity alignments and angular velocities during landing from a single - leg drop vertical jump . the research hypothesis was that landings during fatigue condition decrease the ability to attenuate impact in the lower extremity , i.e. , angular velocities in the lower extremity would increase significantly in landings during fatigue condition compared with non - fatigue condition . thirty - four females ( age : 20.7 1.8 years old ; height : 159.9 5.6 cm ; weight : 52.7 5.9 kg ) without a history of orthopedic hip , knee and ankle surgery participated in this study . the dominant foot was right in 30 subjects and left in 4 subjects ; dominance was determined as preferred side to kick a ball22 . all participants granted written informed consent for participation before testing and were randomly assigned to either the fatigue group ( n=17 ; age : 21.2 2.1 years old ; height : 160.5 5.0 cm ; weight : 52.9 7.1 kg ) or control group ( n=17 ; age : 20.2 1.4 years old ; height : 159.3 6.3 cm ; weight : 52.6 4.5 kg ) . this study followed the declaration of helsinki and was approved by the ethics committee at the saitama medical university , saitama , japan ( m-54 ) . a three - dimensional motion analysis system employing eight cameras ( vicon mx system , vicon motion systems , oxford , uk ) was used to record lower extremity kinematic data and the center of mass during single - leg drop vertical jumps . kinematic data were sampled at 240 hz with a 16 hz low - pass filter and a fourth - order zero lag butterworth filter . thirty - five reflective markers were placed on specific anatomical landmarks according to the plug - in - gait full body model , whisch is widely employed by researchers who use vicon23 . two force plates ( msa-6 mini amp , amti , ma , usa ) recorded ground reaction forces during landing , from each single - leg drop vertical jump , at a 1,200 hz sampling rate . they performed a single - leg drop vertical jump on their dominant foot ( referred to as the pre - trial ) , which involves a first landing after dropping from a 40 cm . a single - leg drop vertical jump consists of 1st landing after dropping down from a 40 cm box and then a second landing after a maximum vertical jump rebounding from the drop . all participants were shown the testing sequence by assistant researchers , and several practice trials were conducted to enable them to correctly perform the required task . subsequent trials were repeated until data from five successful trials were achieved ; these were excluded if the person lost their balance during the landing . after the pre - trial , all participants were required to use a bike ergometer ( combi , japan ) , with participants in the fatigue group pedaling at 100 w per minute for 5 minutes or until they exceeded 17 ( very hard ) on the borg scale24 , 25 . a poster showing the borg scale was placed in front of the bike ergometer , and level of fatigue was recorded at 30 second intervals . in contrast , all participants in the control group pedaled a bike ergometer without load ( less than 10 w per minute ) for 5 minutes , with fatigue level was verified in the same way . after using the bike ergometer , all participants repeated the second single - leg drop vertical jump trial ( referred to as the post - trial ) according to the pre - trial procedure . landing from a single - leg drop vertical jump was defined as the period from initial ground contact to takeoff during the first landing . two force plates were used to determine initial ground contact and takeoff , with the sampling rate set at 1,200 hz . initial ground contact was defined as the moment when the force plate data indicated that vertical ground reaction force exceeded 10 n , whereas takeoff was defined as the moment when the force plate data indicated that vertical ground reaction force was < 10 n. all outcome measures were analyzed and computed as the average of three pre - trials and post - trials . the angles and angular velocities of hip flexion , knee flexion , and ankle dorsiflexion analyzed during all first landings . hip flexion , knee flexion , and ankle dorsiflexion angles were calculated as positive values from filtered three - dimensional coordinate data ; these parameters were defined as angular displacements from static anatomical positions . to decrease data fluctuation , hip flexion , as well as knee flexion , and ankle dorsiflexion angular velocities , were calculated by differentiating respective mean angles over five frames using a moving average . peak hip flexion , knee flexion , and ankle dorsiflexion angles as well as angular velocities were determined from each maximum value during the first landings of the pre - trials and post - trial . the vertical position of the center of mass was calculated from filtered 3d coordinate data during all first landings ; to decrease data fluctuation , vertical position of the center of mass was calculated by differentiating mean vertical position of the center of mass over five frames using a moving average . minimum vertical position and vertical velocity , which were defined as the lowest position and peak velocity in the direction of the ground , were determined from each minimum value during all first landings . the ratios ( % ) of all kinematic variables in the post - trials to those in the pre - trials were calculated . hip flexion , knee flexion , ankle dorsiflexion angle , and angular velocity at specific times were analyzed the moment of the initial ground contact , 40 milliseconds after the initial ground contact , and the peak vertical ground reaction . the moment of 40 milliseconds after the initial ground contact was chosen because some reports had shown acl strain reached its peak value within approximately 40 milliseconds after initial ground contact26 , 27 . data were analyzed using the spss software ( version 19.0 ) , and unpaired t - tests were used to compare changes in lower - extremity kinematics and center of mass between the fatigue and control groups . ratios of the vertical position of the center of mass during the post - trials to each variable during the pre - trials did not differ significantly between the fatigue group ( 99.5 2.4% ; pre - trial 831.3 40.1 mm , post - trial 827.3 47.2 mm ) and the control group ( 100.4 1.4% ; pre - trial 816.7 45.5 mm , post - trial 819.8 46.2 mm ) . ratios of the vertical velocity of the center of mass during the post - trials to each variable during the pre - trials did not differ between the fatigue group ( 100.8 5.5% ; pre - trial 1,966.8 228.9 mm / s , post - trial 1,978.4 229.5 mm / s ) and the control group ( 99.0 4.2% ; pre - trial 1,969.0 175.0 mm / s , post - trial 1,945.7 161.9 findings were similar for the ratios of post - trial of the vertical position and velocity center of mass to their pre - trial counterparts . the ratios of hip flexion , knee flexion , ankle dorsiflexion angles , and angular velocities during the post - trials to each variable during the pre - trials are shown in table 1table 1.peak angles and angular velocities of lower extremity , and ratios of each variable in the post - trials to those in the pre - trialsfatigue group ( n=17)control group ( n=17)pre - trialpost - trial the ratio ( % ) pre - trialpost - trial the ratio ( % ) hip flexionpeak angle41.1 12.641.8 9.341.2 13.3100.1 6.740.6 10.396.6 8.6peak angular velocity216.7 44.6238.9 51.8231.6 42.5107.8 9.7 * 238.4 56.199.6 9.2knee flexionpeak angle60.8 8.260.0 9.960.6 8.199.8 4.960.1 8.7100.7 6.2peak angular velocity419.2 53.4450.6 57.6443.5 52.4106.2 7.3 * 452.2 49.9100.8 6.6ankle dorsiflexionpeak angle30.1 6.929.2 4.930.5 7.2101.2 5.931.0 4.7107.0 13.5peak angular velocity674.6 22.3715.3 42.0655.8 88.898.0 7.6692.0 142.297.2 9.8the ratios ( % ) of each kinematic variable in the post - trials to those in the pre - trials were presented alongside . angles and angular velocities of hip , knee flexion , and ankle dorsiflexion are shown in each ( deg ) and ( deg / s ) . * significant difference between the ratios of fatigue and control groups ( p<0.05 ) .. the ratio of hip flexion angular velocity during the post - trials to that during the pre - trials increased significantly in the fatigue group . furthermore , the ratio of knee flexion angular velocity during the post - trials to that during the pre - trial also increased significantly in the fatigue group ( p<0.05 ) ; however , the ratio of ankle dorsiflexion angular velocity did not differ between groups during either set of trials . in contrast , peak hip flexion , as well as knee flexion and ankle dorsiflexion angles did not alter after the fatigue protocol ; thus , hip and knee flexion angular velocities increased significantly in the fatigue group compared to the control group , whereas all lower extremity joint angles were unchanged . the ratios ( % ) of each kinematic variable in the post - trials to those in the pre - trials were presented alongside . angles and angular velocities of hip , knee flexion , and ankle dorsiflexion are shown in each ( deg ) and ( deg / s ) . * significant difference between the ratios of fatigue and control groups ( p<0.05 ) . the ratios of hip flexion , knee flexion , ankle dorsiflexion angles , and angular velocities during the post - trials to each variable during the pre - trials at the moment of initial ground contact , 40 milliseconds after the initial ground contact , and the peak ground reaction force , are shown in tables 2table 2.angles of lower extremity and ratios of each variable in the post - trials to those in the pre - trials at specific timesfatigue group ( n=17)control group ( n=17)pre - trial ( deg)post - trial ( deg)the ratio ( % ) pre - trial ( deg)post - trial ( deg)the ratio ( % ) initial ground contacthip flexion angle21.7 8.421.4 6.421.0 8.297.8 16.121.0 6.799.2 12.2knee flexion angle18.2 6.318.5 7.117.1 6.094.5 11.818.4 7.4101.0 16.2ankle dorsiflexion angle16.3 6.115.1 4.915.1 5.893.5 15.213.3 4.390.9 20.140 ms after initial ground contacthip flexion angle25.8 9.025.8 7.025.3 8.899.2 13.125.3 7.4109.1 7.4ankle dorsiflexion angle2.7 5.45.4 3.13.5 5.336.1 409.66.3 4.1130.9 75.9the moment of peak ground reaction forcehip flexion angle30.5 8.930.6 7.529.5 9.496.1 7.229.1 7.795.3 5.194.0 23.7the ratios ( % ) of each kinematic variable in the post - trials to those in the pre - trials were presented alongside and 3table 3.angular velocities of lower extremity and ratios of each variable in the post - trials to those in the pre - trials at specific timesfatigue group ( n=17)control group ( n=17)pre - trial ( deg / s)post - trial ( deg / s)the ratio ( % ) pre - trial ( deg / s)post - trial ( deg / s)the ratio ( % ) initial ground contacthip flexion angular velocity95.8 55.9104.6 67.398.0 54.299.7 37.099.8 56.4118.4 13.4ankle dorsiflexion angular velocity600.4 136.1672.5 139.2589.8 106.499.2 6.6642.4 162.195.7 13.140 ms after initial ground contacthip flexion angular velocity186.7 47.8205.0 62.6201.0 48.0109.1 37.0 * 205.9 61.8100.8 8.2knee flexion angular velocity408.3 56.3429.0 65.3425.9 62.1104.4 7.0434.1 57.6101.7 6.3ankle dorsiflexion angular velocity601.9 102.2600.6 99.5583.9 75.197.7 7.5579.3 65.097.5 9.3the moment of peak ground reaction forcehip flexion angular velocity207.9 47.2216.9 63.9228.1 41.8111.4 12.7233.6 53.6114.0 39.6knee flexion angular velocity389.8 59.4387.8 77.6427.7 55.5110.6 10.7432.0 47.9115.5 50.5the ratios ( % ) of each kinematic variable in the post - trials to those in the pre - trials were presented alongside . * significant difference between the ratios of fatigue and control groups ( p<0.05 ) . first , results show that angles , and angular velocities of lower extremity joints upon initial ground contact were not altered by the fatigue protocol . second , 40 milliseconds after the initial ground contact , the ratio of hip flexion angular velocity during the post - trials to each variable during the pre - trials increased significantly in the fatigue group in comparison with the control group ( p<0.05 ) , whereas knee flexion and ankle dorsiflexion angular velocities were unchanged following the fatigue protocol . finally , at the peak vertical ground reaction force , lower extremity joint angles and angular velocities were also unchanged following the fatigue protocol . the ratios ( % ) of each kinematic variable in the post - trials to those in the pre - trials were presented alongside the ratios ( % ) of each kinematic variable in the post - trials to those in the pre - trials were presented alongside . * significant difference between the ratios of fatigue and control groups ( p<0.05 ) the purpose of this study was to reveal influences of fatigue on the lower extremity alignments , and angular velocities , during landing from a single - leg drop vertical jump . one of the key findings of this study is that it is important to select appropriate evaluation parameters and timing during landing for assessing effects of fatigue . lower extremity kinematics and ground reaction force during landings alter under fatigue conditions5 , 6 , 11 , and reveal hip or knee angles , moments , and alignments of lower extremities during landing during fatigue conditions . moreover , modification of these parameters by fatigue may a biomechanical factor leading to lower extremity injuries , including acl injuries . although evidence indicates that fatigue alters lower extremity kinematics , and that fatigue is a risk factor for incurring such injuries , most studies measured hip and knee angles6 , 11 , 19 , or moments5 , during landings . however , lower extremity injuries , including acl injuries , may change lower extremity angles or velocities over time , including the phase of changing direction in sidestepping , or landing from a jump . furthermore , rapid changes in lower extremity alignment occur frequently in many situations during sporting activities , and these findings indicate that measuring angle and angular velocity during fatigue is useful for preventing lower extremity injuries . the results of this study show that peak hip flexion and knee flexion angular velocities increased significantly after the fatigue protocol , suggesting that fatigue decreased capacity to perform deceleration movements in the hip and knee joints during landings from jumps . other investigators have suggested that knee kinematics play an important role in attenuating impact during running or jump landings6 , 28 , 29 ; indeed , these results , indicate that fatigue may induce the knee joint to decrease impact attenuation during landings because knee flexion angular velocity increased significantly after the fatigue protocol . moreover , we showed that hip flexion angular velocity increased significantly during landings after the fatigue protocol , and that fatigue might induce the hip and knee joints to decrease impact attenuation during landings . in contrast , peak ankle dorsiflexion angular velocities did not change after the fatigue protocol ; thus , on the basis of these findings , angular velocity of knee and hip flexion during fatigue conditions should be analyzed to evaluate their capacity for impact attenuation by the lower extremities during landings . hip and knee angular velocities altered by fatigue may be important parameters to measure to prevent knee injuries . in this study , we measured hip flexion , knee flexion , and ankle dorsiflexion angles as well as angular velocities , at the moment of initial ground contact , 40 milliseconds after initial ground contact , and peak ground reaction force . the moment of initial ground contact represents the initial phase when contraction of the lower extremity muscles occurs during the landing phase . furthermore , during the phase between initial ground contact and the peak ground reaction force , the ground reaction force in the vertical direction rapidly increased from the moment of initial ground contact during landings . subsequently , it is likely that the ground reaction force continues to decrease until the takeoff from a vertical jump . for these reasons , fatigue may alter lower extremity kinematics upon initial ground contact with increasing muscle contractions of the lower extremity and the vertical ground reaction force . however , lower extremity kinematic values were unchanged by fatigue condition at the moment of initial ground contact and peak ground reaction force . used video analysis to show that most acl injuries occur approximately 2550 milliseconds after initial ground contact during landings17 , and koga et al . have suggested that it is likely that the majority of acl injuries occur within 40 milliseconds after initial ground contact30 . indeed , other evidence shows that acl is strained during the initial phase of landings , approximately 40 milliseconds after initial ground contact , during landings26 , 27 . according to the results of this study , 40 milliseconds after initial ground contact , hip flexion angular velocity increased significantly after the fatigue protocol , whereas other kinematics variables were unchanged . thus , we conclude that fatigue does not affect knee kinematics , 40 milliseconds after initial ground contact , the point at which most acl injuries are considered to occur . in contract , hip flexion angular velocity was significantly affected by fatigue condition 40 milliseconds after initial ground contact . evidence indicates that increased hip flexion angle during landings is a biomechanical risk factor for acl injuries17 . however , we were unable to demonstrate that fatigue affect hip flexion angle or the position of the center of mass during landings , considering that hip flexion angle and the position of the center of mass did not change after fatigue in this study . furthermore , acl injuries are caused by the combined movement of lower extremity angles in the sagittal plane and their variables in the frontal or horizontal planes , including hip adduction or knee abduction angles15 . therefore , it may be necessary to consider the influence of hip flexion velocity on acl injuries by measuring knee abduction and hip adduction angles or knee external or internal angles , or both . this study shows that fatigue decreased the ability to attenuate impact by increasing angular velocity in the direction of hip and knee flexion during a single - leg jump landing . moreover , 40 milliseconds after initial ground contact , hip flexion angular velocity increased during fatigue , whereas knee and ankle kinematics remained unchanged . these results indicate a requirement to evaluate movement quality over time by measuring peak hip and knee flexion angular velocities during the landing phase during fatigue conditions . in addition , it may be necessary to evaluate these parameters at each peak variable and at specific times such as 40 milliseconds after initial ground contact during the landing phase during fatigue conditions . these findings further suggest that measuring hip and knee angular velocity during landings might be useful for efforts to prevent knee injuries during fatigue conditions . the results of this study indicate that fatigue decreases the ability to attenuate impact by increasing angular velocity in the direction of hip and knee flexion during single - leg jump landing . furthermore , it may be necessary to evaluate knee and hip angular velocities at their peak values and a specific time such as 40 milliseconds after initial ground contact , during the landing phase under fatigue conditions . these findings suggest that measuring hip and knee flexion angular velocities during fatigue conditions may serve as important evaluation parameters to prevent knee injuries , including acl injuries .

[ purpose ] fatigue alters lower extremity landing strategies and decreases the ability to attenuate impact during landing . the purpose of this study was to reveal the influence of fatigue on dynamic alignment and joint angular velocities in the lower extremities during a single leg landing . [ subjects and methods ] the 34 female college students were randomly assigned to either the fatigue or control group . the fatigue group performed single - leg drop vertical jumps before , and after , the fatigue protocol , which was performed using a bike ergometer . lower extremity kinematic data were acquired using a three - dimensional motion analysis system . the ratio of each variable ( % ) , for the pre - fatigue to post - fatigue protocols , were calculated to compare differences between each group . [ results ] peak hip and knee flexion angular velocities increased significantly in the fatigue group compared with the control group . furthermore , hip flexion angular velocity increased significantly between each group at 40 milliseconds after initial ground contact . [ conclusion ] fatigue reduced the ability to attenuate impact by increasing angular velocities in the direction of hip and knee flexion during landings . these findings indicate a requirement to evaluate movement quality over time by measuring hip and knee flexion angular velocities in landings during fatigue conditions .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC3459650

neurological disease is one of the major burdens to healthcare , and in the case of neurodegenerative disorders , the incidence will undoubtedly rise with increased life expectancy . consequently , there has been a concerted effort to understand the genetic basis of neurological disease , particularly in the post - genome era in which large population studies are highlighting new susceptibility loci for further study ( 1,2 ) . the mouse has played a key role in discerning the normal function of genes in the nervous system but also the consequences of their disruption in human disease ( 3,4 ) . mice have a range of advantages as a model system in neuroscience , including their ability to perform behavioural tasks and respond to pharmacological challenges that can be extrapolated directly to human phenotypic traits ( 5,6 ) . the generation of mouse knockouts has been a commonly used approach to study gene disruption in the context of a whole organism . recent years have seen the initiation of an internationally coordinated programme to create es cells carrying conditional null alleles for all protein - coding genes , and this will be a valuable resource for the research community ( 7,8 ) . however , in the majority of neurological disorders , a homozygous loss - of - function mutation does not occur and therefore studying a gene knockout may produce a far more severe phenotype or initiate compensatory mechanisms that are not wholly relevant to the disease in question ( 9 ) . indeed , a large proportion of human disease is caused by mutations that influence protein structure , binding affinity or function in far subtler manner than complete inactivation ( 10,11 ) . in addition , it is becoming apparent that genetic variants such as single nucleotide polymorphisms ( snps ) outside of protein - coding regions are likely to play an important role in disease ( 1,2 ) . alternative mouse models are therefore vital , and random mutagenesis has become a well - established tool to elucidate both gene function and characterize novel disease pathways ( 12 ) . several large - scale mutagenesis programs were established in the late 1990s as a systematic approach to generating new mouse models of human disease ( 1315 ) . the various strategies are reviewed extensively elsewhere ( 16,17 ) , but briefly , male mice are treated with a controlled dose of n - ethyl - n - nitrosourea ( enu ) that causes random point mutations to occur in their germline . mutagenized males are then bred to wild - type females over one or two generations to generate mutant animals for analysis ( 16 ) . in a phenotype - driven approach , the progeny is screened initially for abnormalities using a simple yet quantitative assessment of physiology and behaviour ( 18,19 ) . this is followed by further analysis of specific traits or body systems of interest , as discussed in more detail below ( 14,15,20 ) . inheritance testing of the selected phenotype is then confirmed prior to positional cloning of the mutation itself . one advantage of this random mutagenesis approach is it can create a range of mutations , including hypomorphic ( reduced amount of gene product ) , hypermorphic ( increased amount of gene product ) and neomorphic ( altered function ) alleles , in addition to those that are loss of function , such as the replacement of a coding amino acid with a premature stop codon . the mutation may also be specific for a particular isoform of the gene , which may have important functional consequences . furthermore , the unbiased nature of the phenotyping process and the pleiotropic nature of mutations mean that novel functions can be assigned for genes that had previously only been studied using a restricted set of experiments in the knockout mouse ( see below ) . the major bottle - neck of the phenotype - driven approach used to be the identification of the causative mutation based on genetic mapping followed by sequencing of candidate genes . with the advent of affordable next - generation sequencing technology , this notoriously laborious process can now be completed far more efficiently ( 2123 ) . phenotype - driven screening will not detect every potentially interesting mutant and is limited by the sensitivity of the screens themselves ( 24,25 ) . neuropathological analysis , although a key feature of the phenotyping process , may also be limited to specific tissues or histopathological methods ( 26 ) . nevertheless , cryopreservation of tissues from each mutant line allows retrospective screens to be carried out for mutations in a gene of interest ; advances in the rapid detection of mutations have made it practical to use this gene - driven approach ( 27,28 ) . although there is no guarantee of any measureable phenotype in the resulting mutant , it has been calculated from pilot studies that 5000 dna samples are sufficient to identify at least two alleles with 90% confidence ( 29 ) , and enu archives much larger than this are now available for screening ( 28,30 ) . furthermore , the fact that many thousands of mutants are generated means that multiple individual mice may contain mutations in the same gene , an allelic series ( 31 ) . phenotype correlations to be carried out on mice of the same genetic background which is an important consideration , particularly in behavioural neuroscience ( 6,32 ) . a number of approaches have been taken to quantify behaviour in phenotype - driven mutagenesis screens , and the major large - scale enu programmes have focussed on a range of neurobehavioural domains for their primary screening protocols ( 14,15,20 ) . typically , high - throughput tests such as the open - field are employed as they are both rapid and electronically quantifiable . advantageously , this test provides preliminary data on a number of parameters , including general activity in a novel environment as well as a measure of anxiety based on thigmotaxis ( 33 ) . yet even such deceptively simple tests rely on consistency between those handling the animals and data interpretation to generate scientifically meaningful results ( 34,35 ) . this is paramount when the performance of a single animal compared with a control data set may define a new mutation of importance ; thus , care must be taken to define standardized protocols ( 32,3638 ) . nonetheless , even tests that are used to model aspects of neuropsychiatric disorders can be amenable to a high - throughput screen , such as sensorimotor gating or pre - pulse inhibition ( ppi ) . this particular paradigm quantifies the ability to respond to and filter auditory information , and is one parameter that can be analysed in mice as an endophenotype ; the approximation of a similar response known to be abnormal in schizophrenia ( 5 ) . consequently , ppi and open field , along with rotarod testing for motor function and hotplate for nociception , make up the primary screen of several phenotyping pipelines ( 25,37,39,40 ) . an outlier identified from a high - throughput screen is usually taken through a more focussed and/or complementary series of secondary behavioural tests to more accurately define the phenotype of potential interest . for example , inactivity in the open field may be a consequence of anxiety or simply due to motor dysfunction , arousal or sensory defects ( 6 ) . additional physiological parameters outside of the nervous system must also be taken into account when interpreting such data ; an overtly obese mouse , or one with cardiovascular defects may struggle to run on a rotarod , for instance ( 41,42 ) . another issue to consider is the statistical power of the primary screen used to isolate outliers for follow - up studies ( 20 ) . recent work based on the open - field test has demonstrated that subtler phenotypes can be reliably identified by screening an additional second generation ( g2 ) of mutant animals . although this entails the additional time and cost of more breeding , it was argued that the false discovery rate was dramatically reduced ( 43 ) . despite these caveats , a considerable number of neurological enu mutants have been successfully mapped and cloned , revealing important new insights into gene function in the nervous system ( 16,44 ) . the inherent complexity of the molecular mechanisms that define the function of the brain means that many enu mutants with abnormal neurological phenotypes are yet to be characterized . importantly , primary screening data are usually publically available from the major centres allowing searches to be made for phenotypes of interest [ see box 1 in gondo et al . table 1 contains a selection of the more recently identified neurological enu mutants , and some of those that have been studied in some detail are discussed below , focussing on three key areas of neurological disease research . table 1.examples of neurobehavioural enu mouse mutants ( 2007)mutant gene namemutation(s ) studiedphenotypic effectsreferencesaf4v280a/+ataxia , cataracts , t cell development defects , adult - onset purkinje cell death(52,55,57)arcn1i422t / i422tcoat colour dilution , ataxia , purkinje cell death(128)atp13i810n/+small size , seizures , elevated exploratory locomotion , sleep abnormalities(85,88)cacna1ar1255l/+ataxia , cerebellar atrophy(129)cacna1ar1255l / r1255lsevere ataxia , premature deathdisc1l100p/+ppi , latent inhibition defects(71)disc1l100p / l100pppi , working memory , latent inhibition defects , hyperactivitydisc1q31l/+ppi defectsdisc1q31l / q31lppi , working memory , latent inhibition defects , depressive - like behaviourdisc1q31l / l100pppi defectsfbxl3c358s/+lengthened circadian period(102,109)c358/c358lengthened circadian period , reduced anxiety , depression - like behaviourfoxp2r552h/+normal development , defects in motor learning(130)foxp2r552h / r552hsevere motor impairment , developmental delay , cerebellar foliation defects , 34-week survivalfoxp2s321x / s321xsevere motor impairment , developmental delay , cerebellar foliation defects , 34-week survivalfoxp2n549k / n549klate - onset cerebellar foliation defects , 35-month survivalfoxc1f107l / f107lcortical and skull abnormalities(131)garsc201r/+reduced grip strength , reduction in axonal diameter(63)garsc201r / c201rneurodeveopmental delay , 3-week survivalgrin1c844r/+hyperactivity , increased novelty seeking(132)npc1d1005g/+abnormal cholesterol metabolism , purkinje cell loss , late - onset ataxia(126)ror - alphac257x / c257xataxia , severe cerebellar atrophy(133)rtn4r189h / r189hgreater social preference , subtle cognitive defects(134)snap-25bi67t/+subtle ataxia , ppi defects , advanced circadian phase(76,106)trpc3t635a/+ataxia , late - onset purkinje cell death(59)jenna / tuba1as140g/+hyperactivity , increased acoustic startle , neuronal migration defects(135137)tuba1ad85g/+hyperactivity , neuronal migration defects(138 ) examples of neurobehavioural enu mouse mutants ( 2007 ) historically , spontaneous mouse mutants were identified during the maintenance of large breeding colonies . as a consequence , phenotypes such as ataxia represent many of the first lines to be cloned as such characteristics are particularly conspicuous ( 45,46 ) . the same is true to some extent of phenotype - driven mutagenesis , where there will often be a bias towards movement defects that are both early onset and easy to recognize . for example , three independent enu mutants in the peripheral myelin protein 22 ( pmp22 ) gene were some of the first to be cloned and characterized from the mutagenesis screen at mrc harwell ( 14 ) . initially identified due to their resting tremor , these models of human peripheral neuropathy provided an excellent example of the range of phenotypic and pathological severity that can be generated by an allelic series of point mutants ( 4749 ) . ataxia or defects in motor co - ordination are features of many neurological disorders and are typically characterized by neurodegeneration in the cerebellum , the structure in the brain necessary for controlling motor function . although the genetic cause for a considerable proportion of inherited ataxias is known , the pathogenic mechanisms involved are still unclear ( 50,51 ) . as part of a screen to identify new models of human ataxia , the dominant enu mutant robotic ( rob ) was selected due to its jerky , ataxic gait that is apparent from as early as 3 weeks of age . pathological analysis revealed a progressive yet highly specific loss of cerebellar purkinje cells that begin several weeks after the onset of the ataxic gait ( 52 ) . the causative mutation was subsequently identified in the gene af4 ( all fused from chromosome 4 , also known as aff1 ) that at that time was known to be a putative transcriptional activator implicated in leukaemia ( 53 ) . the mouse knockout of af4 has defects in b- and t - cell development consistent with a role in leukaemogensis , but no neurological abnormalities were reported ( 54 ) ; consequently , the rob mouse revealed an important new function for af4 in the cns that was not apparent from the null allele . biochemical data demonstrated that the mutant protein accumulated in rob purkinje cells due to the loss of a conserved ubiqutin ligase - binding site , and that this increase in af4 protein levels was detrimental to purkinje cell survival ( 55 ) . understanding the proposed gain - of - function mechanism for the rob mutation required detailed examination of the role of af4 in transcriptional regulation . first , it was discovered that af4 and related proteins are part of a large complex co - ordinating rna polymerase ii processivity and chromatin remodelling via histone methylation ( 56 ) . subsequently , transcriptional profiling of purkinje cells from wild - type and rob mice identified igf-1 as target of the af4-containing complex and revealed a pathogenic pathway in the mutant cerebellum ( 57 ) . significantly , dysfunction of the igf-1 pathway is a recurrent finding in mouse ataxic mutants and human ataxia patients and thus represents a common neuropathological mechanism that may be targeted for treatment ( 58 ) . moonwaker ( mwk ) , a second dominant ataxic mutant characterized from the same enu screen , also displays gait defects around weaning age , although purkinje cell loss in this case is later in onset from around 6 months of age ( 59 ) . the causative mutation was identified in the trpc3 gene , encoding the transient receptor potential cation channel c3 ( trpc3 ) . the point mutation occurs in a highly conserved cytoplasmic linker region of trpc3 , resulting in altered gating and increased activity of the channel as a gain - of - function mechanism ( 59 ) . significantly , purkinje cell morphology is impaired in the mwk mouse , which is consistent with the highly specific expression of trpc3 in purkinje cells during their dendritic development . genetic ablation of this channel in the mouse also causes gait defects ( 60 ) ; however , the mwk enu mutant defines a novel role for trpc3 in both the development and survival of purkinje cells . although no proven causative mutations have been described to date ( 61 ) , a polymorphism influencing the methylation of trpc3 was reported in idiopathic ataxia , although its frequency did not reach the significance in the population analysed ( 62 ) . for example , a mouse originally identified due to its reduced grip strength contains a mutation in glycyl t - rna synthetase or gars ( 63 ) . gars heterozygous mutants show a reduction in the axon diameter of peripheral nerves and some muscle denervation , but no axonal degeneration . gars homozygous mutants , however , only survive for a few weeks with major defects in the cns observed . importantly , this gene is mutated in charcot - marie - tooth type 2d ( cmt2d ) disease , a form of dominantly inherited motor neuropathy . the relatively mild phenotype in gars mice differs from the more severe gars dominant mutant that also contains a point mutation ( p279k ) in the same gene ( 64 ) . these mice display an early - onset loss of peripheral nerve axons and abnormal morphology of the neuromuscular junction . interestingly , a gene - trap insertional mutant that is essentially a complete gene knockout shows no dominant phenotype , suggesting that haploinsufficiency is not the cause of neuropathy in the two - point mutants ( 64 ) . to investigate this further , both point mutants were crossed with transgenic lines over - expressing either wild - type or mutant forms of gars . these studies demonstrated that the dominant neuropathy phenotype observed in gars and gars mice is caused predominantly by a dose - dependent gain of function that is not alleviated by over - expression of the wild - type protein ( 65 ) . considering that dominant mutations in three other trna synthetase genes cause neuropathies similar in pathology to cmt2d , these mouse lines will continue to be valuable models of the heterogenous nature of these disorders . the significant genetic component of psychiatric disorders is well - documented and many susceptibility genes are conserved in rodents , although it is accepted that examining specific symptoms related to emotions or psychotic behaviour can not be realistically therefore , research has relied on the aforementioned endophenotypes to analyse specific pathophysiological features of psychiatric disease by studying mutations in genes directly associated with a particular disorder , relevant neurobiological processes such as neurotransmission , or behavioural responses to pharmacological agents known to modulate particular symptoms ( 6,6769 ) . perhaps a reflection of the complex aetiology of psychiatric disease and interpretation of behavioural assays in mice , relatively few enu mutants have been characterized in this area ; however , some important examples have been identified . of all the genes implicated in schizophrenia , disrupted - in - schizophrenia 1 ( disc1 ) is one of the most prominent , with both the disc1 locus and polymorphic variants in the disc1 gene associated with the disorder ( 70 ) . this gene was therefore selected for gene - driven screening in a mouse enu mutant archive , in which two missense mutations were identified ( 71 ) . interestingly , both lines showed a range of behavioural deficits although few were shared ; the disc1-q31l homozygous mutant displayed depressive - like behaviour , whereas the disc1-l100p homozygous line showed abnormalities related to schizophrenia , such as ppi and cognitive defects . these schizophrenic endophenotypes were reversible with antipsychotic treatment ( 71 ) as well as an inhibitor of gsk3 , a known signalling molecule that interacts with disc1 during neurodevelopment ( 72 ) . detailed pathological studies have since identified morphological defects in the dendrites of l100p cortical neurons ( 73 ) . these two mutants show how point mutations in a single gene can cause a heterogeneous behavioural profile . as a caveat to this work , an independent group has been unable to reproduce either the depressive or sensorimotor gating abnormalities in these mutants ( 74 ) ; this could be explained by the additional generations of backcrossing to the wild - type ( c57bl/6 ) strain carried out prior to testing or differences in the behavioural protocols , for example ( 75 ) . the dominant blind - drunk ( bdr ) mutant was originally identified due to its subtle gait disturbance and positional cloning isolated a missense mutation in the neuronal isoform of snap-25 , part of the core snare complex essential for exocytosis ( 76 ) . the mutation causes increased binding affinities within the snare complex and disrupted exocytotic vesicle recycling in vivo , limiting neurotransmission under continued stimulation ( 76 ) . behavioural testing of these mice showed no overt cognitive deficits , but an impaired ppi response . importantly , the study of snare proteins has been limited by the neonatal lethality of knockout lines ( 77,78 ) ; bdr therefore provides valuable new insights into the role of the snare complex and aberrant neurotransmission relevant to psychiatric disease due to the relatively subtle nature of the mutation . furthermore , snap-25 itself has been implicated in schizophrenia from a range of genetic ( 79 ) , pathological ( 80,81 ) and functional ( 82 ) studies of the synapse . bipolar disorder shares some overlapping symptoms with schizophrenia as part of a spectrum of psychotic disorders , although it is associated more readily with both manic and depressive episodes . many genes have been implicated in bipolar disorder , although similar to schizophrenia , it is unlikely that a single genetic factor will be causative ( 83,84 ) . in a phenotype - driven screen , the myshkin ( myk ) dominant mutant was selected due to its small size in addition to the presence of spontaneous , recurrent convulsive seizures and was originally presented as a model of epilepsy ( 85 ) . a missense mutation in the na , k - atpase ( nka ) 3 isoform ( atp1a3 ) caused an approximate 40% reduction of the enzyme activity in the myk brain ; this gene has been implicated both in bipolar disorder and rapid - onset dystonia parkinsonism ( 86,87 ) . when examined in more detail , the behavioural phenotype in myk mice also included highly elevated exploratory locomotion and sleep abnormalities in addition to enhanced sensitivity to d - amphetamine ; significantly , these mania - related behaviours could be attenuated with mood stabilizers such as lithium ( 88 ) . studies of primary neurons suggested that enhanced calcium - dependent signal - transduction pathways downstream of nka may underlie the behavioural phenotypes and are potential therapeutic targets ( 88 ) . myk mice therefore provide important insights into neuronal signalling in mania , particularly as epilepsy and bipolar disorder can be co - morbid in humans ( 89 ) . non - genetic influences are undoubtedly a major risk factor in psychiatric disease , ranging from prenatal stress to adverse life events during adolescence ( 90,91 ) . in recent years , increasing attention has been paid to how best to incorporate such elements into behavioural experiments in mice to understand the synergy between genes and the environment while potentially improving the construct validity of such models ( 9294 ) . building on work in wild - type rodents , there is now a range of gene environment paradigms being applied to mutant mice , and enu lines have provided useful examples of these methods . to examine the significance of early life events combined with a synaptic mutation , a variable prenatal stress protocol was applied to the bdr snap-25 mutant and significantly , the sensorimotor gating ( ppi ) deficits observed in non - stressed mutants were markedly enhanced by stress in utero and social interaction abnormalities were observed only in bdr animals from stressed dams ( 95 ) . these results showed for the first time that combining a synaptic mouse point mutant with a prenatal stressor paradigm produces both modified and previously unseen phenotypes relevant to the study of psychiatric disorders . disc1-l100p and -q31l mutants have also been further studied in the context of a postnatal stress paradigm . chronic social defeat ( csd ) , a correlate of psychosocial stress in humans , was applied to both heterozygous mutant lines and , interestingly , each responded differently to the csd stressor ( 96 ) . measures of anxiety were influenced by csd in the q31l line , whereas social interaction was affected in the l100p mutant ( 96 ) . the exact pathophysiological mechanisms that underlie such gene environment interactions are still unclear , but these studies serve to illustrate that enu mutants with subtle behavioural defects are highly suited for assessing this phenomenon . circadian rhythms are of vital importance to mammalian physiology , and as a consequence , disruption of these daily cycles can have profound effects on human health . indeed , studies are beginning to define a direct association between disrupted rest / activity patterns and an increasing number of neurological disorders , including psychiatric and neurodegenerative disease ( 9799 ) . in the mouse , there are well - established methods for analysing aspects of circadian behaviour , predominantly based around quantifying wheel - running activity under differential lighting conditions ( 100 ) . screening enu mutants using these methods , although not as high - throughput as other behavioural screens , has been particularly successful ; a considerable number of lines displaying abnormally long or short active periods or aberrant responses to shifts in the light / dark cycle have been identified ( 101 ) . of those that have been characterized at a mechanistic level , the afterhours ( afh ) mutant displayed considerably lengthened activity profiles under constant lighting conditions , with a free - running period of approximately 27 h ( 102,103 ) . afh contains a loss - of - function mutation in the e3 ubiquitin ligase fbxl3 , causing a reduction in the normal degradation of cryptochrome ( cry ) proteins . a post - translational feedback loop containing cry and period ( per ) is essential for normal pacemaking in the suprachiasmatic nucleus ( scn ) , and it was shown that the imbalance of normal cry levels resulted in an extended period of the core clock in these mutants ( 102,103 ) . by crossing the afh line to a casein kinase ( csnk1e ) mutant that causes destabilization of per , it was recently shown that the circadian period is finely controlled by both cry and per proteins independently as opposed to the mutual stabilization model predicted by in vitro experiments ( 104 ) . although the mechanisms that link disturbed sleep / wake profiles and psychiatric disease are unclear , it is intuitive that the multiple brain regions disrupted in conditions such as schizophrenia are likely to overlap with those involved in the complex processes of sleep and the maintenance of circadian rhythms ( 105 ) . in view of the schizophrenic endophenotypes in the bdr mouse ( see above ) , this mutant line was also screened for circadian activity ( 106 ) . the behavioural data showed an advanced phase of activity under light / dark cycles , but also fragmentation of the profiles , somewhat reminiscent of the disturbed sleep / wake patterns observed in schizophrenia ( 107 ) . interestingly , the timing of activity was normal under constant light or dark , suggesting that the core clock in bdr was functioning correctly . this was confirmed by molecular analysis of the scn , although expression of the signalling neuropeptide arginine vasopressin ( avp ) was phase advanced in line with the activity data . these results suggest that disrupted neurotransmission in the scn due to the bdr snap-25 mutation influences important hypothalamic outputs , potentially causing desynchrony between the brain and the periphery ( 106 ) . due to the undoubted anatomical overlap between complex behavioural domains , it is also becoming clear that mouse mutants originally identified from circadian screens are likely to show additional behavioural abnormalities ( 108 ) ; indeed , homozygous afh mutants also show hyperactivity , reduced anxiety and depression - like behaviour , suggesting this mutant may also model some aspects of mania related to disrupted circadian rhythms ( 109 ) . the examples above illustrate the complex and varied behavioural phenotypes that can be generated by either phenotype- or genotype - driven enu mutagenesis . another practical application of enu mutants is for identifying new loci that either suppress or enhance a disease phenotype of interest , also known as a modifier screen . successfully utilized in lower organisms ( 110 ) this strategy may be particularly suited to neurodegenerative disease models , where performance in a simple motor - coordination task would provide the baseline data and used to identify outliers in a genetic cross with a population of random mutants ( 16 ) . in particular , the identification of a suppressor mutation may help identify new therapeutic targets or disease mechanisms . a prime example of this phenomenon using a single enu mutant line is the legs - at - odd - angles ( loa ) mouse that contains a mutation in the cytoplasmic dyenin heavy chain ( dync1h1 ) ( 112 ) . while crossing loa mice to model of huntington 's disease enhances the pathological phenotype by slowing clearance of huntingtin protein ( 113 ) , the neurodegenerative phenotype of the sodg93a model of amyotrophic lateral sclerosis ( als ) is significantly suppressed ( 114 ) . preliminary data from an enu screen for modifiers of k3 mice , a model of alzheimer 's disease ( ad ) , have recently been described ( 115 ) . this model expresses mutant tau protein and has been well studied for pathological and behavioural abnormalities ( 116 ) ; consequently , a simple battery of motor tests could be used to define the k3 phenotype . a number of mutant pedigrees , including one that partially rescued the tremor observed in k3 mice , were described , although the identification of this potential suppressor mutation or the analysis of additional cognitive phenotypes was not reported ( 115 ) . such studies therefore rely on a robust and quantifiable phenotypic assay for the disease model in the first instance ; however , this is a potentially very valuable and unbiased approach to understanding how in the interaction between genes can influence pathogenic pathways . while detailed optimization and standardization of enu mutant phenotyping is undoubtedly important , due to obvious practical and financial constraints , primary screening is typically carried out on adult mice between 8 and 18 weeks of age ( 25,37 ) . as a consequence , with particular reference to age - related disorders such as neurodegeneration , potentially valuable mouse models that display late - onset phenotypes or pathology would not be detected . to address this issue , a small number of ageing enu screens have been established , with the aim of testing cohorts of mutants at several time points up to 18 months of age or beyond [ for examples , see http://www.har.mrc.ac.uk/research/functional-genomics-and-disease-mechanisms-section/disease-model-discovery-and-translatio-2 and johnson ( 117 ) ] . it is well - established that the behavioural response in ageing mice can be quite different from younger animals ( 118 ) , and issues of general health and weight must also be taken into account ( 119 ) . however , these challenging and time - consuming studies are a very important way of identify genes and pathways involved in age - related disease . finally , looking further ahead , advances in sequencing technology and data analysis will have an important role to play in the exploitation of enu mutants , not just at the positional cloning stage . gene - driven screens are currently limited to screening small numbers of loci for an allelic series of mutations ; however , it will become financially feasible in the coming years to sequence the entire genome of all archived mutants . another approach would be to focus on the mouse exome or even the transcriptome from archived tissue ( 120 ) . although this would dramatically reduce the amount of data to generate and analyse , with the realization that non - protein - coding sequences also play such a key role in the function of the cns ( 121,122 ) , a genome - wide database of mutants would maximize the potential of the enu mutagenesis resource that numbers over 50 000 individually archived lines . indeed , enu mutations in regulatory non - coding regions causing distinct phenotypes have already been described ( 123,124 ) . as more is revealed regarding the complexity and multi - factorial nature of human neurological disease , access to models that recapitulate aspects of the behavioural and/or pathological abnormalities associated with these disorders is essential . with the ability to carry out longitudinal studies to identify early biomarkers and by focussing on specific endophenotypes , the mouse will continue to be a vital tool to identify disease pathways in neurodegenerative and psychiatric disease research . enu mutagenesis has already generated a significant number of new clinically relevant mouse models , and technological improvements in phenotyping and sequencing strategies will only help improve the efficiency of their identification . access to point mutants will also complement gene knockout studies to model the heterogeneous range of human disease phenotypes . for example , the well - studied mouse model of the lysosomal storage disorder niemann - pick type c ( npc ) does not express any npc protein and serves as a model for the severe infantile forms of this disease ( npc1 ) ( 125 ) ; however , a recently described enu mutant ( npc1 ) provides a more practical model of the more common yet later - onset npc pathology that is caused by missense mutations ( 126 ) . furthermore , the number of potential genetic variants that lead to disease susceptibility is ever - expanding , including novel snps or de novo copy number variants that will drive future mouse modelling experiments ( 127 ) as well as epigenetic and environmental factors . therefore , in combination with transgenics and conditional knockouts , enu mutagenesis will continue to play an important role in the identification and characterization of mutations that underlie human neurological disease .

identifying genes involved in behavioural disorders in man is a challenge as the cause is often multigenic and the phenotype is modulated by environmental cues . mouse mutants are a valuable tool for identifying novel pathways underlying specific neurological phenotypes and exploring the influence both genetic and non - genetic factors . many human variants causing behavioural disorders are not gene deletions but changes in levels of expression or activity of a gene product ; consequently , large - scale mouse enu mutagenesis has the advantage over the study of null mutants in that it generates a range of point mutations that frequently mirror the subtlety and heterogeneity of human genetic lesions . enu mutants have provided novel and clinically relevant functional information on genes that influence many aspects of mammalian behaviour , from neuropsychiatric endophenotypes to circadian rhythms . this review will highlight some of the most important findings that have been made using this method in several key areas of neurological disease research .

INTRODUCTION BEHAVIOURAL SCREENING MOVEMENT DISORDERS AND NEURODEGENERATION NEUROPSYCHIATRIC DISEASE CIRCADIAN BEHAVIOUR FUTURE PROSPECTS CONCLUSION FUNDING

PMC3899023

many biochemical processes in the eukaryotic cell are controlled by the posttranslational modification of proteins with ubiquitin ( ub ) , a 76 amino acid polypeptide that is highly conserved among eukaryotes ( kerscher et al . , 2006 ) . ub adopts a globular structure in which all seven lysine residues and the n terminus are solvent exposed and can therefore form linkages with the c terminus of another ub to form poly - ub chains ( ye et al . , 2012 ) . the ub linkage type and the chain length encode the fate of a ubiquitinated substrate . protein modification by k48-linked poly - ub chains is a well - established signal for recognition and initiation of degradation by the 26s proteasome complex , whereas proteins tagged with k63-linked ub chains are known to be involved in nonproteolytic events such as in dna damage responses and immune signaling ( komander and rape , 2012 ) . the roles of atypical poly - ub chains linked through the ub n terminus , k6 , k11 , k27 , k29 , or k33 are less well understood , but proteins tagged in such a way also appear to be directed toward proteasomal degradation ( kulathu and komander , 2012 ; xu et al . , 2009 ) . the molecular basis for the biological diversity of poly - ub chains stems from their distinct structural features . some ub - linkages such as k6 , k11 , and k48 adopt compact structures where adjacent moieties interact with each other whereas linear and k63-linked chains display more open conformations where the linkage site is the only interface , although all of them show a degree of flexibility and dynamics ( ye et al . , 2012 ) . this implies that enzymes involved in ubiquitin conjugation ( e1/e2/e3 ligases ) and deconjugation ( deubiquitinating enzymes [ dubs ] ) must bear specific recognition features to create and process different poly - ub chains in their biological context , often involving direct interactions with cognate protein substrates and perhaps with other components of multiprotein complexes they are embedded in ( reyes - turcu et al . , 2009 ) . dubs are proteases that mediate the removal or processing of ub chains or linked ubl proteins by hydrolyzing the isopeptide bond between the c - terminal glycine of the distal molecule and the lysine residue in either the proximal ub or the protein substrate . dubs have recently emerged as key regulators in a multitude of processes from gene transcription to protein degradation and from cell division to cell death ( nijman et al . , 2005 ; tsou et al . , they consist of a protease family comprising 90 members in human cells that can be grouped into five distinct families including ubiquitin - specific proteases ( usps ) , ubiquitin c - terminal hydrolases ( uchs ) , ovarian tumor domain proteases ( otus ) , and machado - joseph disease ( mjd ) protein family , all of which are cysteine proteases . available knowledge of dub specificity or mode of regulation in cells is scant due to challenges in isolating dub containing protein complexes or expressing full - length recombinant enzymes ( reyes - turcu et al . , 2009 ) . current efforts to study dubs in vitro generally focus on the production of recombinant enzymes and the use of artificial substrates ( e.g. , ub - amc ) . such investigations may not always reflect their substrate specificity and selectivity in a cellular environment . although k48- and k63-linked ubiquitin dimers and oligomers have been accessible through chemoenzymatic synthesis for a number of years ( pickart and raasi , 2005 ; piotrowski et al . , 1997 ) , synthetic access to the less well studied atypical linkages has only been established more recently . selectively functionalized ub variants have been assembled from peptide building blocks using native chemical ligation - based strategies ( kumar et al . , 2010 ; yang et al . , 2010 ) as well as by optimized solid phase peptide synthesis of full length ub ( el oualid et al . , 2010 ) . in each of these strategies , a mercapto - lysine residue was utilized for ligation with a c - terminal ub thioester followed by desulfurization to obtain an isopeptide linkage containing a native lysine residue . genetic incorporation of protected boc - lysine in escherichia coli followed by orthogonal amine protection schemes and ag(i)-mediated thioester to the successful genetic incorporation of protected mercapto - lysine derivatives in e. coli has now also enabled the combination of unnatural amino acid incorporation with the facile ligation methodology enabled by mercapto - lysine derivatives ( virdee et al . , 2011 ) . in addition to methods for the generation of native isopeptide - linked ub dimers , there has also been an increased interest in novel straightforward methods for ligation of ub by non - native linkages . cu(i)-catalyzed triazole formation has been successfully employed for the generation of ub - protein conjugates ( weikart and mootz , 2010 ) as well as ub dimers ( eger et al . , 2010 ; it was further demonstrated that the obtained conjugates are resistant to cleavage by dubs ( weikart and mootz , 2010 ) . as an addition to the repertoire of conventional in vitro assays , ub - based active site probes , targeting dub catalytic sites in crude cell extracts were successfully used to discover and profile active enzyme species ( borodovsky et al . , 2001 ; de jong et al . , 2012 ; love et al . , 2009 ) and small molecule dub inhibitors ( altun et al . , 2011 ) . the latter approach does not require biochemical purification of dubs and can report on their activity and inhibition within their biological context , but so far was restricted to single ub or ub linked to a branched peptide as recognition scaffolds ( iphfer et al . , 2012 ) . in this study , we have engineered active site probes on a dimeric ubiquitin ( di - ub ) scaffold representing all eight different ub - linkages , using biochemical methods coupled with discovery proteomics and quantitative mass spectrometry to define dub poly - ub linkage specificity in a cellular environment . activity - based di - ub probes formed of two recombinant ub proteins were designed ( figure 1 ) with the following consideration in mind : the isopeptide bond present in the natural ub dimer was to be replaced by a reactive electrophile for the covalent capture of cysteine protease dubs . the linkage position was controlled by site - directed mutagenesis and incorporation of the unnatural amino acid azidohomoalanine ( aha ) into the proximal ub using the methionine analog incorporation approach ( kiick et al . , 2002 ) . subsequently , the proximal ub was linked by cu(i)-catalyzed 1,4-triazole formation to the c terminus of the distal ub . molecular modeling indicated similarities in spatial requirements of the linker region between the natural linkage containing a gly to lys isopeptide bond and the reactive electrophilie linked via a 1,4-triazole ( figure s1 available online ) . a recent computational study using quantum mechanical / molecular mechanical ( qm / mm ) methods confirmed that k48-linked ub - dimer contains similar geometric features in both native ub dimer and a synthetic triazole - linked ub dimer ( dresselhaus et al . , 2013 ) . the comparison was carried out between k48-linked ub dimer with a native isopeptide linkage and a triazole containing dimer based on click conjugation of a k48azphe containing ub . with this approach , di - ub probes mimicking linkages to each of the seven lysine residues or the n - terminal methionine were synthesized . the ha - tag present on the n - terminal of the distal ub ( derived from ha - ub(1 - 75)-alkyne ) allowed visualization and retrieval for identification using tandem mass spectrometry ( lc - ms / ms ) . introduction of an alanine residue at position two of ubiquitin ( i2a ) to facilitate n - terminal met / aha cleavage by methionine aminopeptidase ( wang et al . , 2008 ) was followed by individual k to m point mutations at each of the lysine residues to afford the seven ub mutant plasmids . the ub mutants and wt ub were expressed in a methionine auxotrophic e. coli strain for azidohomoalanine incorporation ( m1aha , k6aha , k11aha , k27aha , k29aha , k33aha , k48aha , k63aha ) and purified ( pickart and raasi , 2005 ) . maldi - tof ms of the intact proteins displayed efficient n - terminal met / aha cleavage as determined by the strength of signal at 8,504 m / z ( corresponding to the mass of n - terminal cleaved ub ) versus a negligible to minor signal detected at 8,639 m / z ( corresponding to uncleaved ub ) . lc - ms / ms analysis after digestion with trypsin was also carried out to verify site - specific aha incorporation ( see figures 2 , s2 , and dna sequences for wild - type and mutant ubiquitin ) . ha - ub - alkyne and vme probes were prepared and purified according to literature procedures ( borodovsky et al . , 2001 , 2002 ; mcgouran et al . , 2012 ) the ha - ub - alkyne was ligated to the relevant ub aha mutant by cu(i ) catalyzed triazole formation ( rostovtsev et al . , 2002 ) to afford the corresponding ha - tagged di - ub probes . hplc purification of all di - ub probes was performed using strong cation exchange chromatography to obtain high purity preparations removing any unconjugated ha - ub - alkyne . the separation of ub material of hplc fractions was monitored by sds - page and anti - ha western blotting ( figure 3a ) . the fractions marked with a dotted box were pooled , concentrated , and used for subsequent ub processing enzyme profiling experiments . representative hplc chromatograms and intact protein maldi - tof ms spectra are shown for the m1 di - ub probe ( figures 3b and 3c ) . the reactivity of the purified m1 di - ub probe was tested by incubation with the deubiquitinating enzyme otubain 2 ( otub2 ) alongside the monomeric ha - ub - alkyne probe and visualized by both silver stain and western blotting ( figure 4a ) . covalent trapping of otub2 with the probes was observed as a characteristic mass shift and shown to be inhibited by the cysteine alkylating agent n - ethylmaleimide ( nem ) . in order to demonstrate the requirement for the presence of a reactive electrophile , a proportion of the material was deactivated by reaction with sodium 2-mercaptoethanesulfonate ( mesna ) . inactivated and active m1 di - ub probes were incubated with four different recombinant dubs ( otub2 , uch - l3 , uch - l5 , and usp15 ) and also a cellular extract prepared from hek293 t cells ( figure 4b ) . all dubs were successfully labeled with the active m1 di - ub probe , but did not react with the inactivated counterpart . incubating the active probe with lysate resulted in strong labeling of proteins whereas lysate incubation with the inactive probe did not lead to any labeling . this demonstrates that the electrophilic trap is required for covalent capture of cellular dubs ( figure 4b ) . furthermore , conjugation through the native c terminus of the proximal ub of the di - ub probe by e3 ligases does not occur to any significant extent under these labeling conditions . to verify site - specific linkage of the ubiquitin moieties within the di - ub probes , lc - ms / ms analyses after digestion with trypsin were performed ( figures 5a and s3a s3f ) . to assess the effect of introducing a nonnative triazole linker on the conformation of di - ubiquitin , surface plasmon resonance ( spr ) was carried out to compare the k48 di - ub probe to wild - type ( wt ) k48 di - ubiquitin with regards to its binding affinity for otub1 , a dub selective for k48-linked poly - ub chains ( edelmann et al . , 2009 ) ( figure 5b , left and middle panel ) . a catalytically inactive mutant ( otub1 c91s ) was used to avoid covalent binding of the probe . spr analysis indicated kd values of 50 nm ( di - ub wt ) versus 80 nm ( di - ub probe ) , respectively ( figure 5b , right panel ) . the otub1 inactivation mutation may further stabilize the substrate interaction as compared to its wild - type enzyme ( wiener et al . , 2013 ) , but appears to be similar between natural di - ub and the di - ub probe . in order to compare differences in the cellular labeling pattern of the eight di - ub probes , the ha - ub - vme and ha - ub - alkyne probes , the respective probe and hek293 t cell lysate were incubated and analyzed by sds - page and anti - ha immunoblotting ( figure 6 ) . interestingly , di - ub probe labeling profiles are distinct from one another and notably so from the mono - ub probes , suggesting a higher dub selectivity . to investigate this in more detail , cell lysate probe labeling followed by immunoprecipitation ( ip ) with anti - ha antibody agarose beads the elution fractions of all ips were analyzed by tandem mass spectrometry to identify ubiquitin processing enzymes trapped by the probes . in total , 29 dubs were identified by lc - ms / ms in all ten probe ips combined including 18 usps , four uchs , five otus , one mjd , and one sumo deconjugase ( figure 7a ) . the mass spectrometric intensities of peptide signals for detected dubs in all experiments were subjected to relative quantitation in order to compare dub selectivity for the various ub linkages . in separate experiments , poly - ub chains of defined linkage were incubated in vitro with various recombinant members of the uch , usp and otu families to compare and further explore dub preferences for different ub linkages . samples were analyzed by sds - page and anti - ub immunoblotting to visualize disassembly of the respective ub chains ( figures 7b7i ) . as a positive control all recombinant dubs were labeled with ha - ub - vme , demonstrating that all enzymes used in this panel were active ( figure 7j ) . the monomeric ha - ub - vme and alkyne probes showed generally the lowest labeling specificity across the panel of cellular dubs detected ( figure 6 , lanes 1 and 2 ) , whereas interestingly , the m1 di - ub probe was the one with the most promiscuous specificity among the di - ub probes ( figure 7 , lane 3 ) . it should be noted that although the di - ub probes display the electrophilic trap in the appropriate position to mimic the native isopeptide bond ( figures 1 and s1 ) , the m1 probe is linked via the amino acid side chain ( m1 position ) rather than the protein n terminus . the different relative position of the distal ubiquitin and the greater degree of flexibility afforded by this side chain linkage in comparison to the native peptide bond apparently cause decreased specificity of this probe ( komander et al . , 2009 ) . although many dubs showed little reactivity toward the different di - ub linkage types in comparison to the mono - ub probes , several were selective for particular di - ub probes tested . we detected all four cysteine protease members of the uch family in our probe ip experiments . consistent with the observation that uch enzymes are inefficient at ubiquitin chain cleavage ( kulathu and komander , 2012 ; zhou et al . , 2012 ) , we observed low general reactivity toward the di - ub probes in comparison to the monomeric probes in our ip samples ( figure 7a ) . we also detected little evidence of cleavage in our recombinant enzyme assays ( figure 7b ) . with otu6b , we observe little reactivity toward the di - ub probes in comparison to the monomeric probes . 2010 ) , does indeed reflect this specificity in our assay showing the highest reactivity among the di - ub probes , although the specificity we observe is somewhat more modest than that seen within the previously reported in vitro assays with recombinant enzymes ( mevissen et al . , 2013 ) . otub1 displayed k48 linkage - specificity among the di - ub probes ( figure 7a ) , in line with both literature observations ( edelmann et al . 2010 ) and results of our poly - ub cleavage assay ( figure 7 g ) . vcip interestingly displayed a preference for the k11 linkage among the di - ub probes in line with in vitro assays using truncated vcip ( mevissen et al . , 2013 ) , although little activity was seen with the k48 di - ub linked probe as would be expected from the in vitro assays using the truncated form ( mevissen et al . , 2013 ) . for usps , it has previously been noted that these enzymes are generally specific to ubiquitination substrates rather than the linkage of the ubiquitin chain , with a low degree of selectivity observed between the various ubiquitin linkages ( faesen et al . , 2011 ) . our poly - ub cleavage assays and probe labeling experiments confirm this observation , although we do see some examples of apparent specificity in the reactivity toward different di - ub probes . for instance , usp13 , usp25 , and usp40 showed high relative affinity for the m1 probe . interestingly , previous in vitro assays suggested little reactivity with linear ub chains for these dubs ( faesen et al . , 2011 ; kulathu and komander , 2012 ) . this discrepancy could be due to either the artificial nature of the in vitro assay or the greater flexibility of the linkage present in our m1 di - ub probe as compared to the native peptide bond ( komander et al . , 2009 ) . interestingly , otulin , a dub exclusively specific for cleaving linear poly - ub chains ( keusekotten et al . , 2013 ) , was not detected with the m1 di - ub probe , possibly due to similar reasons . usp15 in our ip results showed a degree of selectivity for k27-linked probe ( figure 7a ) . the corresponding poly - ub cleavage assay showed that usp15 in its recombinant form was indeed active in cleaving k27 chains , but also other linkages ( figures 7b7i ) . usp16 showed a high relative reactivity toward the k27 and to a lesser extent the k29 probe in our assay . literature precedence suggests that usp16 is active against these chain types although a less striking selectivity was observed previously ( faesen et al . , 2011 ) . di - ub probe labeling data also suggested k11 selectivity by usp5 ( figure 7a ) , whereas our poly - ub cleavage data suggested a very low degree of reactivity generally ( figures 7b7i ) . previous data has , however , shown usp5 activity against k11 chains ( bremm et al . , 2010 ) . usp47 was found to have a high degree of specificity toward k27 linkages based on reactivity toward the di - ub probes and the recombinant dub assays , and usp24 was highlighted to have a high degree of specificity toward the k27 and k11 probes . we also detected ataxin 3 , principally in the m1 and k29 probe ips despite previous suggestions that it has low activity toward chains of four ubs or less ( mao et al . ub fusion protein 1 was also detected , and the highest relative abundance was observed with the m1 di - ub probe . brca1-a showed little selectivity among the different probes . taken together , our engineered probes representing all naturally occurring di - ub linkages provide clues on topology preferences of cellular dubs for different poly - ub chains , some of which were validated in vitro . interestingly , many dubs appear also to recognize noncanonical ub - linkages other than k48/63 scaffold , suggesting the processing of a wider range of ubiquitinated substrates with different ub linkage topologies . quantitative mass spectrometry analysis of di - ub probe ip material also revealed components of the ub conjugation machinery were enriched by the different di - ub probes , as has been previously observed with mono - ub probes ( love et al . , 2009 ) . a panel of conjugating enzymes was detected including those from hect and rbr e3 families along with ring e3 ligases and e2 family members ( table s2 ) . the application of engineered activity probes based on di - ub scaffolds allows us to address dub ub - linkage specificity in a cellular context , which generally appears less biased toward canonical ub - linkages than previously anticipated . certain dubs , in particular otus , retain selectivity for distinct poly - ub chains as predicted using recombinant enzyme assays . the approach described provides insights into the selectivity of dubs within the cognate environment of substrates as part of multiprotein complexes , which is often problematic to reconstitute with in vitro experiments . the detection of many enzymes of the ubiquitin conjugation machinery is noteworthy and may also provide us with a useful tool to study such enzymes in a cellular environment . these advances provide a framework for further understanding the role of dubs and ub conjugating enzymes in cellular functions under normal physiological conditions as well as in disease . a greater understanding of dub ub - linkage specificity may also offer inroads to assess cellular effects and specificity of small molecule inhibitors for future pharmacological intervention strategies . reagents were purchased from aldrich and used as supplied . deionized water ( minimum resistivity 18.2 m ) was used for preparation of buffers and chemical and biochemical reactions . precision plus protein standard all blue gel marker ( biorad ) was used for sds - page . ha - ub - alkyne , ha - ub - vme probes , and azidohomoalanine were synthesized in house according to literature procedures ( borodovsky et al . , 2002 ; link et al . , 2007 ; mcgouran et al . , 2012 ) using a ha - ub(1 - 75)-intein - chitin t cells were cultured in dmem ( dulbecco ) medium supplemented with 10% fcs ( sigma ) and 1% penicillin / streptomycin at 37c with 5% co2 . following overnight culture bacteria were harvested and the wild - type plasmid dna containing the ubiquitin sequence cloned into the pet15b ( addgene ) vector was isolated , using wizard plus sv minipreps dna purification system ( promega ) according to the manufacturer s protocol . dna concentration was analyzed by measuring absorption at 260 nm wavelength using a nanodrop spectrophotometer . point mutants of ubiquitin were created using the quickchange ii site directed mutagenesis kit by stratagene . mutant i2a was used as template for all following substitutions : k6 m , k11 m , k27 m , k29 m , k33 m , k48 m , and k63 m . the primers used for the design of the ubiquitin mutants are listed in table s1 . mutant constructs were verified by dna sequencing ( source bioscience , department of biochemistry , university of oxford ) . sequencing results can be found in the dna sequences for wild - type and mutant ubiquitin . for the expression of wt and mutant ub , methionine auxotroph b834(de3 ) competent cells were used ( novagen ) according to manufacturers protocol . glycerol stocks were prepared by picking a single colony from an agar plate and incubating it in lb medium with shaking ( 160 rpm ) at 37c for 18 hr . a 100 l cell suspension was mixed with 900 l of 60% glycerol in water and stored at 80c . bacteria were incubated in selenomet medium ( molecular dimensions ) containing 40 g / ml methionine at 37c for 18 hr . after transferring the bacteria to up to 500 ml selenomet medium containing and methionine , bacteria were grown with shaking ( 180 rpm ) at 37c until they reached an od600 of 0.9 . after harvesting , the cells were washed two times with methionine - free selenomet medium before resuspension in selenomet medium containing 40 g / ml azidohomoalanine ( aha ) and 0.4 mm iptg . protein expression was accomplished at 37c for 4 hr with continuous shaking ( 180 rpm ) . subsequently the harvested bacteria were resuspended in lysis buffer ( 100 mm tris ph 7.5 , protease inhibitors [ roche ] , 0.02% np40 , 0.4 mg / ml lysozyme ) before 1.2 mg / ml mgso4 and 12 g / ml dnasei were added . vigorously vortexed cell lysate was cleared by centrifugation ( 16,000 g , 30 min , 4c ) and treated with 0.5% perchloric acid on ice . remaining ub in the supernatant was dialyzed in 50 mm naoac ph 4.5 for 18 hr at 4c . the ub mutants were resolved on an 18% bis - tris gel and visualized by silver staining . linear mode maldi - tof ms analysis ( bruker ) was also performed ( figure 2 ) . a further aliquot of each mutant was subjected to digestion by trypsin and the resulting peptides analyzed by lc - ms / ms ( mackeen et al . , 2010 ) to confirm aha incorporation . in brief , proteins were resuspended in 6 m urea , reduced with dithiothreitol and alkylated with iodoacetamide before proteolysis with trypsin . desalted samples were separated on a 75 m 250 mm reversed phase nano - acquity - uplc ( waters ) and analyzed by a coupled q - tof premier mass spectrometer ( waters ) . ms / ms spectra were searched against a human database containing the ub mutant sequences ( 86,755 sequences ) in mascot ( matrix science v2.4 ) allowing one missed cleavage and 50 ppm/0.1 da mass deviation in ms and ms / ms , respectively . oxidation of methionine , methionine to azidohomoalanine exchange , and deamidation of asparagine and glutamine were used as variable modifications . ms / ms spectra showing incorporation of aha in the positions m1 , k6 , k11 , k27 , k29 , k33 , k48 , and k63 are shown in figures s2a s2h . hplc - purified ha - ub - alkyne ( 77 l , 2 mg / ml ) was mixed with the appropriate azidohomoalanine incorporated ub mutant ( 805 l , 0.56 mg / ml ) in 50 mm na2hpo4/nah2po4 ph 8 buffer . tris[(1-ethoxycarbonylmethyl-1h-1,2,3-triazol-4-yl)-methyl]amine ( 90 g in 15 l ch3cn ) and cu(i)br ( 120 g in 15 l ch3cn ) were added before shaking at room temperature . after 30 min , a second portion of cu(i)br ( 120 g in 15 l ch3cn ) was added , prior to a further 30 min incubation with shaking . the di - ub probe was buffer exchanged into 50 mm naoac ph 4.5 using gel filtration ( pd-10 , ge healthcare ) . the resulting probes were hplc ( agilent 1100 ) purified using a scx biomonolith column ( 5.2 4.95 mm , agilent ) with a linear gradient from 0% to 100% buffer b , 50 mm naoac ( ph 4.5 , buffer a ) , 50 mm naoac ( ph 4.5 ) , and 1 m nacl ( buffer b ) at a flow rate of 0.5 ml / min . product - containing fractions were identified based on sds - page separation and anti - ha immunoblotting ( figure 3 ) and concentrated . the correct linkage was confirmed by lc - ms / ms analysis for seven out of the eight di - ub probes ( figures 5a and s3a s3f ) . a minority of purifications , most notably for k33 di - ub , contained tri - ub species possibly resulting from insufficient cleavage of the n - terminal azidohomoalanine leading to double ligation . these by - products were removed . one hundred nanograms of recombinant dubs otub1 , otub2 , uch - l1 , uch - l3 , uch - l5 , bap1 , usp2 , usp5 , usp7 , usp8 , usp15 , usp20 , usp21 , usp28 , and usp47 ( all obtained from progenra ) or 20 g hek293 t cell lysate , prepared as described ( mcgouran et al . , labeling was conducted in presence or absence of 10 mm n - ethylmaleimide ( nem ) to confirm specificity . volumes were adjusted to 15 l with 50 mm nah2po4/na2hpo4 ph 8.0 buffer . all reactions were incubated at 37c for 3 hr followed by addition of 5 l of 3 reducing sample buffer and heating to 95c for 5 min . samples were resolved on a nupage 4%12% bis - tris gradient gel and visualized by silver staining or western blotting methods using a directly coupled anti ha - hrp antibody ( 1:10,000 dilution , sigma aldrich h6533 ) . an inactivated linear di - ub probe was also generated to discount the possibility of conjugation through the di - ub c terminus . two micrograms of linear di - ub probe was incubated in 10 l inactivation buffer ( 50 mm mesna , 50 mm nah2po4/na2hpo4 ph 8.0 ) at 37c for 24 hr . a total of 1.5 l of 50 mm mesna was added along with 10 l 50 mm nah2po4/na2hpo4 ph 8.0 and di - ub probe was incubated for a further 40 hr at room temperature ( rt ) . the buffer was exchanged to 50 mm nah2po4/na2hpo4 ph 8.0 by centrifugal filter devices . alternatively , 1 m recombinant dubs were incubated with 100 ng of poly - linked ub chains ( k6/k11/k27/k29/k33/k48 , or k63 , biomol ) for 3 hr at 37c in 50 mm tris ( ph 8.0 ) and 1 mm dtt , separated by nupage 4%12% bis - tris gradient gel electrophoresis , and analyzed by anti - ub immunoblotting ( 1:3,000 dilution , bd pharmingen ) . surface plasmon resonance experiments were performed on a biacore 2000 instrument equipped with research grade cm5 sensor chips ( biacore ab ) . all surfaces were prepared by an amine - coupling method ( johnsson et al . , 1991 ) using reagents available from biacore ab ( n - ethyl - n-dimethylaminopropyl - carbodiimide [ edc ] , n - hydroxysuccinimide [ nhs ] , and 1 m ethanolamine - hcl ) . flow cells were activated for 15 min at 5 l / min with a solution containing 50 mm nhs and 0.2 mm edc . k48 di - ub wt and k48 di - ub probe were diluted to 20 g / ml in 10 mm sodium acetate ( ph 4.5 ) and injected until immobilization levels of 12,00014,000 ru were achieved . the remaining reactive groups were blocked with a 12 min injection of 1 m ethanolamine - hcl at ph 8.5 . otub1 ( c91s mutant ) binding data were collected at 20c at a flow rate of 5 l / min in a solution of running buffer ( 10 mm hepes [ ph 7.4 ] , 150 mm nacl , 3 mm edta , and 0.005% p20 ) for both reference and reaction surfaces . otub1 c91s was dissolved directly in the running buffer at a stock concentration of 100 m . serial dilutions were made in running buffer across the concentration series from 10 nm to 3 m . samples were injected for 300 s followed by re - equilibration with running buffer . the raw response data were zeroed on both the response and time axes prior to the start of the injection . to correct for bulk refractive index changes , responses from a reference surface without protein were subtracted from the reaction surface data . to extract equilibrium dissociation constants , we plotted binding responses at equilibrium against ligand concentration and measured concentration values at 50% site occupation ( figure 5b ) . preparation of cell lysate and labeling of endogenous active dubs using the ha - ub - alkyne , ha - ub - vme , and ha - di - ub probes was performed according to literature procedures ( borodovsky et al . , 2002 ) . for the quantitative mass spectrometry experiments , 1 mg of hek293 t cell lysate was incubated with 20 g of di - ub probe ( or 10 g of the mono - ub probe ) in net buffer ( 50 mm tris ph 7.5 , 5 mm edta , 150 mm nacl , 0.5% np-40 ) in a total volume of 180 l for 3 hr at 37c . sds was then added to 0.5% final concentration and the protein mixture was incubated at 4c for 1 hr . the sds concentration was reduced to 0.05% by adding net buffer before the mixture was added to monoclonal anti - ha agarose beads ( sigma aldrich ) . immunoprecipitation was carried out at 4c for 18 hr followed by three washing steps with high salt net - buffer ( 50 mm tris ph 7.5 , 5 mm edta , 300 mm nacl , 0.5% np-40 ) . elution was accomplished by addition of ha peptide to a final concentration of 3 mg / ml . the immunoprecipitated eluate was desalted using chloroform - methanol extraction , followed by overnight in - solution tryptic digestion and subsequent desalting as described previously ( altun et al . , 2011 ) . in brief , proteins were chloroform - methanol precipitated prior to resuspension in 6 m urea , reduced with dithiothreitol , and alkylated with iodoacetamide before proteolysis with trypsin . tryptic digests were desalted and subjected to lc - ms / ms ( 75 m 250 mm reversed phase nano - acquity - uplc , waters ) analysis using a thermo ltq orbitrap velos ( 30,000 resolution , top 20 , collision - induced dissociation ) workflow and a gradient of 1%40% acetonitrile in 48 min at a flow rate of 250 nl / min . peptides were detected and quantified with progenesis lc - ms software ( version 3.1.4003.30577 ) using default settings ( no deconvolution / deisotoping , 200 most intense ms / ms peaks ) . a merged peak list generated by progenesis lc - ms was searched against the uniprot human database ( version.3.80 , 20,339 entries ) using mascot v2.4 , allowing one missed cleavage and 20 ppm/ 0.5 da mass deviations in ms and ms / ms . oxidation of methionine and deamidation of asparagine and glutamine were used as variable modifications . for label - free protein quantitation raw protein abundance values from progenesis lc - ms were first normalized using quantile normalization and technical replicates aggregated by taking the median normalized value for each protein in each sample . quantitation of enzyme abundance based on the different probe immunoprecipitations was based on at least two unique peptides , confidence score > 50 and anova ( p value ) < 0.05 in order to generate the heat maps for figure 7a .

summaryposttranslational modification with ubiquitin ( ub ) controls many cellular processes , and aberrant ubiquitination can contribute to cancer , immunopathology , and neurodegeneration . the versatility arises from the ability of ub to form polymer chains with eight distinct linkages via lysine side chains and the n terminus . in this study , we engineered di - ub probes mimicking all eight different poly - ub linkages and profiled the deubiquitinating enzyme ( dub ) selectivity for recognizing di - ub moieties in cellular extracts . mass spectrometric profiling revealed that most dubs examined have broad selectivity , whereas a subset displays a clear preference for recognizing noncanonical over k48/k63 ub linkages . our results expand knowledge of ub processing enzyme functions in cellular contexts that currently depends largely on using recombinant enzymes and substrates .

Introduction Results and Discussion Significance Experimental Procedures

PMC2858203

four groups ( 12 each ) of healthy normal - weight ( bmi of 21.524.4 kg / m , aged 2547 years ) subjects ingested either 75 g (= 300 calories ) of glucose ( glucola drink ; fisher scientific , pittsburgh , pa ) , 33 g (= 300 calories ) of cream ( gourmet heavy whipping cream ; land o'lakes , arden hills , mn ) , an equicaloric amount of orange juice , or 300 ml of water after an overnight fast . the content of the dairy cream used includes 70% saturated fat , 28% unsaturated fat , < 2% protein , and no carbohydrates . blood samples were collected before and at 1 , 3 , and 5 h after glucose , cream , or water intake . the human research committee of the state university of new york at buffalo approved this protocol . we had used orange juice obtained from a local supermarket and had used portions of the juice from - or 1-gallon packages for multiple experiments . to minimize any potential for instability of orange juice constituents , we used packages of recently produced not from concentrate mncs isolated and nf-b dna binding activity was measured as described previously ( 1 ) . total rna isolation and rt - pcr for socs3 and tlr inflammatory cytokines was performed as described previously ( 1,8 ) . the specificity and the size of the pcr products were tested by adding a melt curve at the end of the amplifications and by running it on a 2% agarose gel . all values were normalized to expression of three housekeeping genes ( -actin , cyclophilin , and ubiquitin ) . mnc total cell lysates were prepared , and electrophoresis and immunoblotting were performed as described before ( 12 ) . polyclonal or monoclonal antibodies against tlr-2 ( imgenex , san diego , ca ) , socs3 and tlr-4 ( abcam , cambridge , ma ) , and actin ( santa cruz biotechnology , santa cruz , ca ) were used , and all values were corrected for loading to actin levels . insulin , glucose , and free fatty acids ( ffas ) were measured as described previously ( 1 ) . plasma endotoxin concentration was measured by a commercially available kit ( cambrex limulus amebocyte lysate kit ; lonza , walkersville , md ) as described previously ( 1 ) . lipopolysaccharide - binding protein ( lbp ) was measured using an immunoassay kit from cell sciences ( canton , ma ) . dilutions comparable to the amounts ingested from cream , glucose , and orange juice were prepared in plasma or endotoxin - free water . plasma was used as a diluent to maintain a testing medium similar to that in the postchallenge lps measurements . lps concentrations were then measured as described using a limulus amebocyte lysate assay , and endotoxin was calculated ( n = 3 each ) . statistical analysis was performed using sigmastat software ( systat software , san jose , ca ) . statistical analysis of changes from baselines was performed using holm - sidak one - way repeated - measures anova ( rmanova ) . the dunnett two - factor rmanova method was used for all multiple comparisons between different groups . mncs isolated and nf-b dna binding activity was measured as described previously ( 1 ) . total rna isolation and rt - pcr for socs3 and tlr inflammatory cytokines was performed as described previously ( 1,8 ) . the specificity and the size of the pcr products were tested by adding a melt curve at the end of the amplifications and by running it on a 2% agarose gel . all values were normalized to expression of three housekeeping genes ( -actin , cyclophilin , and ubiquitin ) . mnc total cell lysates were prepared , and electrophoresis and immunoblotting were performed as described before ( 12 ) . polyclonal or monoclonal antibodies against tlr-2 ( imgenex , san diego , ca ) , socs3 and tlr-4 ( abcam , cambridge , ma ) , and actin ( santa cruz biotechnology , santa cruz , ca ) were used , and all values were corrected for loading to actin levels . insulin , glucose , and free fatty acids ( ffas ) were measured as described previously ( 1 ) . plasma endotoxin concentration was measured by a commercially available kit ( cambrex limulus amebocyte lysate kit ; lonza , walkersville , md ) as described previously ( 1 ) . lipopolysaccharide - binding protein ( lbp ) was measured using an immunoassay kit from cell sciences ( canton , ma ) . dilutions comparable to the amounts ingested from cream , glucose , and orange juice were prepared in plasma or endotoxin - free water . plasma was used as a diluent to maintain a testing medium similar to that in the postchallenge lps measurements . lps concentrations were then measured as described using a limulus amebocyte lysate assay , and endotoxin was calculated ( n = 3 each ) . statistical analysis was performed using sigmastat software ( systat software , san jose , ca ) . statistical analysis of changes from baselines was performed using holm - sidak one - way repeated - measures anova ( rmanova ) . the dunnett two - factor rmanova method was used for all multiple comparisons between different groups . there was no significant difference in fasting plasma glucose , insulin , or ffas between the groups . there was a significant increase in glucose ( from 80 2 to 110 9 mg / dl , p < 0.01 ) at 1 h after glucose intake , whereas there was no significant change in glucose concentrations after orange juice , cream , or water . 0.001 ) after glucose and orange juice intake , respectively , but did not change significantly after cream or water intake . lipid concentrations did not change after glucose , orange juice , or water intake , whereas the intake of 33 g ( 300 calories ) cream caused a significant increase in the plasma concentration of ffas ( from 0.32 0.09 to 0.60 0.14 mmol / l ) , triglycerides ( from 103 59 to 171 60 mg / dl ) , and vldl cholesterol ( from 19.5 13 to 32.3 15 mg / dl at 3 h ; p < 0.01 for all ) . there was no significant change in the concentration of total cholesterol , ldl cholesterol , and hdl cholesterol after cream . the intake of glucose caused a significant increase in mrna expression of socs3 in mncs ( by 77 22 , 58 16 , and 54 17% over the baseline at 1 , 3 , and 5 h after glucose intake , respectively ( p < 0.02 ) ( fig . 1a ) . there was a significant increase in the mrna expression of socs3 after cream intake by 45 14 and 56 15% over the baseline at 3 and 5 h , respectively ( p = 0.014 ) ( fig . 1a ) , whereas there was no significant change in socs3 mrna after orange juice or water intake . socs1 and socs7 mrna expression did not change significantly after any challenge ( data not shown ) . socs3 protein in mncs also increased significantly after the intake of glucose and cream by 45 16 and 53 18% over the baseline , respectively , but not after orange juice or water ( p < 0.05 ) ( fig . 1c and d ) . change in socs-3 ( a , c , and d ) and tlr-4 ( b , c , and e ) mrna and protein expression in mncs from normal subjects after a 300-calorie drink of cream ( ) , glucose ( ) , orange juice ( oj , ) , or water ( ) . + , p < 0.05 with rmanova comparing changes in relation to baseline after cream and glucose challenges ; # and $ , p < 0.05 with two - way rmanova for comparisons of cream and glucose changes , respectively , to water ( n = 12 ) . the intake of glucose did not result in any change in tlr-4 expression , whereas cream intake induced a significant increase in tlr-4 mrna expression by 37 11% over the baseline ( p < 0.05 ) ( fig . similarly , there was a significant increase in tlr-4 protein levels in mncs after cream intake by 38 18% over the baseline but not after glucose , orange juice , or water intake ( p < 0.05 ) ( fig . there was no significant change in tlr-2 mrna or protein expression after any of the macronutrient challenges ( data not shown ) . tnf- mrna expression in mncs increased significantly by 53 16% at 1 h and 51 10% at 3 h over the baseline after glucose and cream intake , respectively ( p < 0.05 ) ( fig . there was also a significant increase in il-1 mrna expression in mncs after glucose and cream intake by 96 25% at 5 h and 168 32% at 3 h over the baseline , respectively ( p < 0.01 ) ( fig . on the other hand , il-6 expression did not alter significantly after glucose or cream intake ( data not shown ) . there was no increase in the expression of any of these cytokines after orange juice or water . change in tnf- ( a ) and il-1 ( b ) mrna expression in mncs from normal subjects after a 300-calorie drink of cream ( ) , glucose ( ) , orange juice ( oj , ) , or water ( ) . data are means sem . * and + , p < 0.05 with rmanova comparing changes in relation to baseline after cream and glucose challenges ; # and $ , p < 0.05 with two - way rmanova for comparisons of cream and glucose changes , respectively , to water ( n = 12 ) . dna binding by nf-b increased significantly by 53 17 and 54 18% over the baseline ( p < 0.05 ) ( fig . 3a ) at 3 h after glucose and cream intake , respectively . there was no significant change in nf-b binding activity after orange juice or water intake . binding activity in mnc ( a ) and plasma endotoxin concentrations ( b ) in normal subjects after a 300-calorie drink of cream ( ) , glucose ( ) , orange juice ( oj , ) , or water ( ) . data are means sem . * and + , p < 0.05 with rmanova comparing changes in relation to baseline after cream and glucose challenges ; # and $ , p < 0.05 with two - way rmanova for comparisons of cream and glucose changes , respectively , to water ( n = 12 ) . plasma endotoxin concentrations increased significantly after the intake of cream from 0.29 0.03 to 0.41 0.07 endotoxin units ( eu)/ml at 3 h ( 45 17% over the baseline , p < 0.05 ) ( fig . the concentration of lps in plasma after the intake of cream continued to be significantly higher than basal levels up to 5 h. endotoxin contents of cream and orange juice drinks were 104 24 and 85 21 eu / ml , respectively . this was equivalent to a total endotoxin load of 10,400 eu for the cream taken and 55,250 eu for the orange juice drink . considering that the amount of endotoxin ingested is extremely small ( < 5 g ) in comparison to a total estimated amount of 1 g in the gut , it is unlikely that the lps content in drinks contributes significantly to the increase in plasma endotoxin concentrations . the lbp concentration increased significantly after the intake of glucose by 16% over the baseline at 5 h ( from 9.4 0.8 to 11 1.2 g / ml , p < 0.0.5 ) but not after cream or orange juice . there was no significant difference in fasting plasma glucose , insulin , or ffas between the groups . there was a significant increase in glucose ( from 80 2 to 110 9 mg / dl , p < 0.01 ) at 1 h after glucose intake , whereas there was no significant change in glucose concentrations after orange juice , cream , or water . 0.001 ) after glucose and orange juice intake , respectively , but did not change significantly after cream or water intake . lipid concentrations did not change after glucose , orange juice , or water intake , whereas the intake of 33 g ( 300 calories ) cream caused a significant increase in the plasma concentration of ffas ( from 0.32 0.09 to 0.60 0.14 mmol / l ) , triglycerides ( from 103 59 to 171 60 mg / dl ) , and vldl cholesterol ( from 19.5 13 to 32.3 15 mg / dl at 3 h ; p < 0.01 for all ) . there was no significant change in the concentration of total cholesterol , ldl cholesterol , and hdl cholesterol after cream . the intake of glucose caused a significant increase in mrna expression of socs3 in mncs ( by 77 22 , 58 16 , and 54 17% over the baseline at 1 , 3 , and 5 h after glucose intake , respectively ( p < 0.02 ) ( fig . there was a significant increase in the mrna expression of socs3 after cream intake by 45 14 and 56 15% over the baseline at 3 and 5 h , respectively ( p = 0.014 ) ( fig . 1a ) , whereas there was no significant change in socs3 mrna after orange juice or water intake . socs1 and socs7 mrna expression did not change significantly after any challenge ( data not shown ) . socs3 protein in mncs also increased significantly after the intake of glucose and cream by 45 16 and 53 18% over the baseline , respectively , but not after orange juice or water ( p < 0.05 ) ( fig . 1c and d ) . change in socs-3 ( a , c , and d ) and tlr-4 ( b , c , and e ) mrna and protein expression in mncs from normal subjects after a 300-calorie drink of cream ( ) , glucose ( ) , orange juice ( oj , ) , or water ( ) . data are means sem . * and + , p < 0.05 with rmanova comparing changes in relation to baseline after cream and glucose challenges ; # and $ , p < 0.05 with two - way rmanova for comparisons of cream and glucose changes , respectively , to water ( n = 12 ) . the intake of glucose did not result in any change in tlr-4 expression , whereas cream intake induced a significant increase in tlr-4 mrna expression by 37 11% over the baseline ( p < 0.05 ) ( fig . similarly , there was a significant increase in tlr-4 protein levels in mncs after cream intake by 38 18% over the baseline but not after glucose , orange juice , or water intake ( p < 0.05 ) ( fig . there was no significant change in tlr-2 mrna or protein expression after any of the macronutrient challenges ( data not shown ) . tnf- mrna expression in mncs increased significantly by 53 16% at 1 h and 51 10% at 3 h over the baseline after glucose and cream intake , respectively ( p < 0.05 ) ( fig . 2a ) . there was also a significant increase in il-1 mrna expression in mncs after glucose and cream intake by 96 25% at 5 h and 168 32% at 3 h over the baseline , respectively ( p < 0.01 ) ( fig . there was no increase in the expression of any of these cytokines after orange juice or water . change in tnf- ( a ) and il-1 ( b ) mrna expression in mncs from normal subjects after a 300-calorie drink of cream ( ) , glucose ( ) , orange juice ( oj , ) , or water ( ) . data are means sem . * and + , p < 0.05 with rmanova comparing changes in relation to baseline after cream and glucose challenges ; # and $ , p < 0.05 with two - way rmanova for comparisons of cream and glucose changes , respectively , to water ( n = 12 ) . dna binding by nf-b increased significantly by 53 17 and 54 18% over the baseline ( p < 0.05 ) ( fig . 3a ) at 3 h after glucose and cream intake , respectively . there was no significant change in nf-b binding activity after orange juice or water intake . binding activity in mnc ( a ) and plasma endotoxin concentrations ( b ) in normal subjects after a 300-calorie drink of cream ( ) , glucose ( ) , orange juice ( oj , ) , or water ( ) . data are means sem . * and + , p < 0.05 with rmanova comparing changes in relation to baseline after cream and glucose challenges ; # and $ , p < 0.05 with two - way rmanova for comparisons of cream and glucose changes , respectively , to water ( n = 12 ) . plasma endotoxin concentrations increased significantly after the intake of cream from 0.29 0.03 to 0.41 0.07 endotoxin units ( eu)/ml at 3 h ( 45 17% over the baseline , p < 0.05 ) ( fig . the concentration of lps in plasma after the intake of cream continued to be significantly higher than basal levels up to 5 h. endotoxin contents of cream and orange juice drinks were 104 24 and 85 21 eu / ml , respectively . this was equivalent to a total endotoxin load of 10,400 eu for the cream taken and 55,250 eu for the orange juice drink . considering that the amount of endotoxin ingested is extremely small ( < 5 g ) in comparison to a total estimated amount of 1 g in the gut , it is unlikely that the lps content in drinks contributes significantly to the increase in plasma endotoxin concentrations . the lbp concentration increased significantly after the intake of glucose by 16% over the baseline at 5 h ( from 9.4 0.8 to 11 1.2 g / ml , p < 0.0.5 ) but not after cream or orange juice . our data show clearly for the first time that the intake of either 33 g cream or 75 g glucose resulted in a significant increase in the expression of socs3 mrna in the circulating mncs . with cream , this increase was observed at 1 h and continued until at least 5 h , at which time the experiment ended . glucose caused an increase in socs3 , which peaked at 1 h ( 83% over baseline ) and was still elevated at 5 h. in parallel with the induction of socs3 , there occurred an increase in nf-b binding , consistent with an acute inflammatory response in mncs . on the other hand , whereas the intake of cream resulted in an increase in both plasma lps concentration and tlr-4 expression , glucose had no effect on either tlr-4 expression or plasma lps concentration . orange juice did not induce either socs3 or tlrs nor did it induce an inflammatory response . it would thus appear that the oxidative stress and inflammation - inducing actions of the hfhc meal are due to the combination of saturated fat and the carbohydrate ( glucose ) at least . on the other hand , only cream induced an increase in plasma lps concentration and an increase in the expression of tlr-4 , the receptor for lps . thus , saturated fats may have a more profound role in the pathogenesis of postprandial inflammation , as they may also perpetuate inflammation through the increases in lps and tlr-4 . by implication , saturated fats also appear to increase the permeability of intestinal epithelium and contribute to the breakdown of the intestinal barrier . both carbohydrate and saturated fat induced an increase in the expression of socs3 , the mediator of interference in insulin signal transduction . in contrast , orange juice does not induce any of the changes in oxidative and inflammatory stress or an increase in either lps or tlr-4 or socs3 . by implication , it may not affect the intestinal barrier either , because there is no increase in plasma lps concentration after orange juice , despite it containing concentrations of endotoxin similar to those of cream . it is possible that the observed effect of orange juice is attributable to its flavonoids , naringenin and hesperidin , because they exert reactive oxygen species suppressive ( 17 ) and anti - inflammatory effects . these potent effects have been demonstrated in experimental animal models in relation to endotoxin - induced inflammation and in cells in vitro ( 18,19 ) . in this context , it is important to state that our recent data demonstrate that the consumption of orange juice with an hfhc meal prevents not only oxidative stress and inflammation but also the increase in lps concentrations and the increase in the expression of tlr-4 and socs3 observed after ingestion of the hfhc meal alone ( 20 ) . thus , the increase in lbp after an hfhc meal observed by us was probably due to the carbohydrate component of the meal . socs3 has been shown previously to be induced by tnf- , il-6 , and il-1 and is thus a product of inflammation . therefore , it is of interest that mrnas for tnf- and il-1 were induced by glucose and cream intake in parallel with the increase in nf-b binding . however , it was surprising that il-6 was not induced by either glucose or cream . socs3 , as its name suggests , was initially discovered as a molecule that interferes with cytokine signaling . it has since been shown to also interfere with both insulin and leptin signal transduction ( 4,21 ) . the induction of socs3 by cream and glucose intake in combination with the fact that its expression is increased in obese individuals suggests the possibility that chronic excessive fat and carbohydrate intake may result in a chronic increase in socs3 expression . it is also of interest that the intake of such macronutrients may result in resistance to leptin , one of the major signals that promotes satiety and thus potentially reduces food intake . similarly , it is intriguing that the intake of a macronutrient should cause the induction of a molecule ( socs3 ) that would interfere with the signal transduction of insulin , a hormone , which causes the assimilation of nutrients after a meal , including the distribution and storage of fat , carbohydrates , and proteins . because the intake of a modest amount of proinflammatory macronutrients led to the induction of socs3 , which induces concomitant insulin and leptin resistance , our observation raises the issue about the search for foods that are not likely to induce socs3 or inflammation . although socs1 and socs7 genes are also induced by inflammatory cytokines and have been reported to modulate insulin signaling in vitro and in experimental animal models ( 2,22 ) , there are no data about their relevance to human insulin resistance . indeed , their expression did not change after glucose and cream intake . first , it can bind to the -subunit of the insulin receptor ( ir- ) , reduce tyrosine phosphorylation of ir- , and prevent the docking of insulin receptor substrate ( irs)-1 to the receptor ( 23 ) . second , it may bind to irs-1 , and by so doing , it may facilitate its ubiquitination and proteasomal degradation ( 2 ) . by binding to irs-1 , it may also prevent the binding of irs-1 to the p85 subunit of phosphatidylinositol 3-kinase and thus prevent insulin signaling . we have recently shown that socs3 expression is increased in obese individuals and is inversely related to the tyrosine phosphorylation of the ir- and that it is directly related to insulin resistance ( homa - ir ) , bmi , and other indexes of inflammation ( 6 ) . our current observations are consistent with its potential role as a mediator of insulin resistance in the obese . socs3 also interferes with leptin signal transduction by reducing the phosphorylation of the leptin receptor and janus kinase ( 24 ) attached to the leptin receptor . this results in a reduction of phosphorylation of signal transducer and activator of transcription ( stat ) and thus the dimerization of stat . thus , the necessary gene transcription in response to the leptin signal can not occur ( 5 ) . the increase in socs3 expression and the presence of leptin resistance in the obese and their potential reversal with macronutrient restriction have important implications . it is of interest that although the intake of cream induced an increase in the expression of tlr-4 and in plasma lps concentrations , glucose had no effect . because tlr-4 is the specific receptor for lps , it would be of interest to examine the effects of repeated intake of cream as reflected in these indexes and the overall magnitude of the inflammatory process . ( 25 ) reported that high glucose concentrations upregulated tlr-4 expression and its downstream signaling in a monocytic cell line . however , the excursions of glucose concentrations in normal and even obese subjects do not achieve those concentrations postprandially . our investigation deals with postprandial changes in normal subjects . as far as the effects of cream intake are concerned , the amount taken is modest , and the increase in triglycerides is consistent with that in previous studies . it should , therefore , be noted that although the amounts of cream and glucose taken induced similar increases in reactive oxygen species generation and nf-b binding , glucose was not able to induce an increase in the expression of tlr-4 . in summary , the intake of a modest amount of glucose or cream results in a significant induction of socs3 mrna and protein in parallel with the induction of an inflammatory response characterized by an increase in nf-b binding in mncs and the induction of two of the cytokines , tnf- and il-1 , which are known to induce socs3 in experimental animals . in addition , the intake of cream but not of glucose also induces an increase in the expression of tlr-4 mrna and protein while also inducing an increase in plasma lps concentrations . both socs3 and tlr-4 are putative mediators of insulin resistance . in contrast , orange juice intake does not induce oxidative stress , inflammation , socs3 , tlr-4 , or an increase in plasma lps concentrations .

objectivewe have recently shown that a high - fat high - carbohydrate ( hfhc ) meal induces an increase in plasma concentrations of endotoxin ( lipopolysaccharide [ lps ] ) and the expression of toll - like receptor-4 ( tlr-4 ) and suppresser of cytokine signaling-3 ( socs3 ) in mononuclear cells ( mncs ) in addition to oxidative stress and cellular inflammation . saturated fat and carbohydrates , components of the hfhc meal , known to induce oxidative stress and inflammation , also induce an increase in lps , tlr-4 , and socs3.research design and methodsfasting normal subjects were given 300-calorie drinks of either glucose , saturated fat as cream , orange juice , or only water to ingest . blood samples were obtained at 0 , 1 , 3 , and 5 h for analysis.resultsindexes of inflammation including nuclear factor-b ( nf-b ) binding , and the expression of socs3 , tumor necrosis factor- ( tnf- ) , and interleukin ( il)-1 in mncs , increased significantly after glucose and cream intake , but tlr-4 expression and plasma lps concentrations increased only after cream intake . the intake of orange juice or water did not induce any change in any of the indexes measured.conclusionsalthough both glucose and cream induce nf-b binding and an increase in the expression of socs3 , tnf- , and il-1 in mncs , only cream caused an increase in lps concentration and tlr-4 expression . equicaloric amounts of orange juice or water did not induce a change in any of these indexes . these changes are relevant to the pathogenesis of atherosclerosis and insulin resistance .

RESEARCH DESIGN AND METHODS MNC isolation and NF-B DNA binding activity Total RNA isolation and real-time RT-PCR Western blotting Measurement of plasma glucose, insulin, and free fatty acid concentrations Plasma endotoxin and lipopolysaccharide-binding protein concentrations Determination of LPS content in food challenges Statistical analysis RESULTS Plasma glucose, insulin, and lipid concentrations Effect of glucose, orange juice, and cream intake on SOCS3 expression Effect of glucose, orange juice, and cream intake on TLR-4 expression Effect of glucose, orange juice, and cream intake on proinflammatory cytokine expression Effect of glucose, orange juice, and cream intake on NF-B DNA binding Effect of glucose, orange juice, and cream intake on plasma LPS and LBP concentrations CONCLUSIONS

PMC4389134

neonates , especially very low - birth - weight infants , are at high risk for acute kidney injury ( aki ) , and aki is an independent predictor of morbidity and mortality in this patient population [ 15 ] . when used in neonates , renal supportive therapy ( rst ) the decision to offer rst to the smallest of patients is complicated by a number of challenges including limited availability of appropriately sized dialysis catheters , lack of machines designed for neonates , technical difficulties associated with access placement in small infants , as well as individual physician practices , biases and beliefs . limited available literature documenting the use of peritoneal dialysis ( pd ) in extremely low - birth - weight ( elbw ) infants describes uniformly high rates of technical complications ( 2560% ) and mortality ( > 50% ) [ 68 ] . for these reasons , practitioners may defer rst in favor of diuretic therapy and nutritional restriction in elbw neonates or offer no dialytic support at all . we report the successful provision of pd to an 830-g premature infant with oligo - anuric aki and deteriorating respiratory status using a commercially available temporary pd catheter . the patient was a male infant born at 28 and 3/7 weeks gestation weighing 830 g. he was twin b of a monochorionic twin gestation , and the donor in a twin twin transfusion syndrome . the pregnancy was complicated by oligohydramnios , worsening cardiac function and hydrops in twin a necessitating preterm delivery via cesarean section . twin b 's apgar scores were 8 and 8 , at 1 and 5 min , respectively ; however , he developed respiratory distress and required intubation . metolazone was added , but he remained anuric . a renal ultrasound demonstrated echogenic kidneys with normal doppler flow and an empty bladder . by dol 3 , the infant 's weight had risen to 1050 g , a 27% increase from birth weight . laboratory studies were significant for serum sodium 128 meq / l ( reference range , 135145 meq / l ) , serum creatinine 2.3 mg / dl ( reference range , 0.20.9 mg / dl ) and blood urea nitrogen ( bun ) 69 mg / dl ( reference range , 1020 mg / dl ) . the infant was transferred to the university of iowa children 's hospital neonatal intensive care unit for respiratory and renal management . total fluids were restricted to a volume of 75 ml / kg / day for the first 24 h post - transfer with further restriction to 60 ml / kg / day and 1 g / kg / day of protein on dol 45 due to worsening fluid overload and significant decline in respiratory status . the infant 's renal parameters indicated severe anuric renal failure , with a bun and creatinine of 87 and 2.7 mg / dl , respectively . on dol 5 , the infant 's weight was 1096 g , indicating a 32% fluid weight gain from birth . given the dire clinical picture and the likelihood of death without intervention , the neonatology , pediatric surgery and pediatric nephrology teams reviewed the risks and benefits of rst with the patient 's family and devised a pd plan for the infant . the primary technical challenge for initiating pd in this elbw infant was that standard cuffed pd catheters , with lengths from 31 to 39 cm , are too large for an elbw infant 's small abdominal domain . acute pd catheters with 8 cm catheter lengths represent the smallest commercially available pd catheter . however , these are likely still too large for an 800-g infant when placed in the standard fashion via an infra - umbilical stab incision with the catheter directed into the pelvis . to use a true pd catheter rather than improvised equipment ( e.g. large - bore intravenous cannula ) that would be prone to complications , we thus considered a novel placement approach using an acute pd catheter inserted into the left upper quadrant . an 8.5-french , 8-cm commercial temporary pd catheter ( pediatric peritoneal dialysis set cook medical ) was placed at the bedside by a pediatric surgeon . using the seldinger technique , the pd catheter was exchanged for the dilator and directed into the pelvis such that the catheter remained straight . a purse - string suture was used to secure a watertight closure of the posterior rectus sheath . the subcutaneous tissues and skin were closed with running suture to prevent leakage of peritoneal fluid and permit initiation of pd immediately . the exit site was closed around the catheter and dressed . catheter placement was confirmed radiographically ( see figure 1a and b ) and with successful instillation and aspiration of saline from the catheter . see figure 2 for photograph of catheter location . 1.(a ) ap x - ray demonstrating pd catheter placement coursing from the left upper quadrant into the left paracolic gutter . ( b ) lateral x - ray demonstrating pd catheter placement coursing from the left upper quadrant into the left paracolic gutter . ( a ) ap x - ray demonstrating pd catheter placement coursing from the left upper quadrant into the left paracolic gutter . ( b ) lateral x - ray demonstrating pd catheter placement coursing from the left upper quadrant into the left paracolic gutter . following catheter placement , manual pd using the dialy - nate system was initiated with 16 ml volume dwells ( 20 ml / kg based on birth weight ) of 45 min using 1.5% dianeal for 24 cycles per day . the infant developed hypotension , so dwell times were decreased to 30 min followed by 25-min drain periods , resulting in better blood pressures while maintaining adequate ultrafiltration and clearance . within 72 h of initiating pd , the infant achieved a net ultrafiltration of 46 ml / pass . with successful dialysis , after 3 days , total fluid intake was liberalized to 120 ml / kg / day for venous nutrition . weight decreased to 1024 g from a peak of 1092 g over the course of 10 days . the infant was transitioned from high frequency to conventional ventilation on dol 13 . by dol 15 , urine output ml / kg / hr , and the infant maintained appropriate fluid balance during trials off pd . renal labs improved prior to discharge to a bun of 28 mg / dl and a serum creatinine of 0.9 mg / dl . he is being followed by pediatric nephrology for the monitoring of his chronic kidney disease . current laboratory studies demonstrate a serum creatinine of 0.5 mg / dl at age 16 months , suggesting an estimated glomerular filtration rate of 61 ml / min/1.73 m by the modified schwartz equation . the primary technical challenge for initiating pd in this elbw infant was that standard cuffed pd catheters , with lengths from 31 to 39 cm , are too large for an elbw infant 's small abdominal domain . acute pd catheters with 8 cm catheter lengths represent the smallest commercially available pd catheter . however , these are likely still too large for an 800-g infant when placed in the standard fashion via an infra - umbilical stab incision with the catheter directed into the pelvis . to use a true pd catheter rather than improvised equipment ( e.g. large - bore intravenous cannula ) that would be prone to complications , we thus considered a novel placement approach using an acute pd catheter inserted into the left upper quadrant . an 8.5-french , 8-cm commercial temporary pd catheter ( pediatric peritoneal dialysis set cook medical ) was placed at the bedside by a pediatric surgeon . a small stab incision was made in the left upper quadrant . a dilator was then placed through the stab incision into the abdomen . using the seldinger technique , the pd catheter was exchanged for the dilator and directed into the pelvis such that the catheter remained straight . a purse - string suture was used to secure a watertight closure of the posterior rectus sheath . the subcutaneous tissues and skin were closed with running suture to prevent leakage of peritoneal fluid and permit initiation of pd immediately . catheter placement was confirmed radiographically ( see figure 1a and b ) and with successful instillation and aspiration of saline from the catheter . see figure 2 for photograph of catheter location . 1.(a ) ap x - ray demonstrating pd catheter placement coursing from the left upper quadrant into the left paracolic gutter . ( b ) lateral x - ray demonstrating pd catheter placement coursing from the left upper quadrant into the left paracolic gutter . ( a ) ap x - ray demonstrating pd catheter placement coursing from the left upper quadrant into the left paracolic gutter . ( b ) lateral x - ray demonstrating pd catheter placement coursing from the left upper quadrant into the left paracolic gutter . photograph of pd catheter placement . following catheter placement , manual pd using the dialy - nate system was initiated with 16 ml volume dwells ( 20 ml / kg based on birth weight ) of 45 min using 1.5% dianeal for 24 cycles per day . the infant developed hypotension , so dwell times were decreased to 30 min followed by 25-min drain periods , resulting in better blood pressures while maintaining adequate ultrafiltration and clearance . within 72 h of initiating pd , the infant achieved a net ultrafiltration of 46 ml / pass . with successful dialysis , after 3 days , total fluid intake was liberalized to 120 ml / kg / day for venous nutrition . weight decreased to 1024 g from a peak of 1092 g over the course of 10 days . the infant was transitioned from high frequency to conventional ventilation on dol 13 . by dol 15 , urine output had improved to 34 ml / kg / hr , and the infant maintained appropriate fluid balance during trials off pd . renal labs improved prior to discharge to a bun of 28 mg / dl and a serum creatinine of 0.9 mg / dl . he is being followed by pediatric nephrology for the monitoring of his chronic kidney disease . current laboratory studies demonstrate a serum creatinine of 0.5 mg / dl at age 16 months , suggesting an estimated glomerular filtration rate of 61 ml / min/1.73 m by the modified schwartz equation . to our knowledge , our case report includes the smallest infant with whom pd was successful using a standard , commercially available pd catheter . rst was indicated given severe oligo - anuric aki resistant to medical management , fluid overload resulting in respiratory and hemodynamic compromise and inability to provide adequate nutrition secondary to fluid restriction . use of rst in neonates is often limited by lack of size - specific equipment . pd was chosen for rst due to the lack of appropriately sized vascular catheters for a patient of such small size . due to our infant 's extremely small size and severe fluid overload , his initial dialysis prescription also required adaptation with increased initial fill volumes of 20 ml / kg to overcome the comparatively large amount of dead space within dialysis tubing ( a catheter volume of 0.4 ml and a connecting tube volume of 3.0 ml ) . limited existing literature documents high mortality rates in elbw infants treated with pd , though the high rates in some reports are likely related to the underlying medical conditions . for example , two case series using pd in neonates each reported > 50% mortality but included many infants with inborn errors of metabolism [ 6 , 7 ] . continuous veno - venous hemodialysis is the preferred therapy for patients with this condition because of its superior efficacy ; inefficient clearance with pd is not adequate for rapid removal of ammonia in these cases . similarly , one group reported a 75% mortality rate in the use of pd for infants with necrotizing enterocolitis , a condition associated with high mortality rates as well as one which would be considered a relative contraindication to the use of pd because of the inflammation in the abdomen and high risk for infection . in addition to high mortality rates , the available literature describes high rates of complications such as catheter leak and peritonitis during the use of pd in small infants , likely related to the lack of size - specific equipment for these patients . use of varying catheter types , both commercial and improvised , for pd in elbw neonates has been described . two larger case series of pd in neonates ( bw range , 14 kg ) report technical success using rigid catheters in the infra - umbilical position [ 6 , 7 ] . the patients in these series describe successful use of a flexible 10-french blake silicone drain in a 40-week term infant requiring pd . detail novel use of a vascular catheter placed in the right upper quadrant ; however , high rates of catheter leak , poor drainage , and kinking were noted . similarly , use of a large - bore intravenous cannula has been described ; however , again this improvised device was prone to malfunction with leak and kinking . to our knowledge , this case illustrates the first time a standard , temporary pd catheter has been placed successfully via a left upper abdominal quadrant insertion site facilitating uncoiled extension into the left paracolic gutter in an elbw infant . early initiation of rst for aki and fluid overload in neonates may have important short- and long - term systemic effects . adult and experimental data suggest that mortality due to aki may be related to multi - organ functional and transcriptional changes , especially in the lungs [ 1619 ] . aki is implicated in the development of bronchopulmonary dysplasia due to pulmonary edema from volume overload and the associated elevation in pro - inflammatory cytokines . the adult critical care literature demonstrates that critically ill patients with aki are more difficult to wean from mechanical ventilation . these findings bolster the need for early fluid removal and treatment of aki in neonates given the implications for sequelae of chronic lung disease in this vulnerable population . herein , the neonatology team was able to transition our infant from high frequency to conventional ventilation within 1 week of dialysis initiation . our infant 's severely fluid restricted state prior to rst initiation eliminated any opportunity to provide nutrition without further fluid accumulation . adequate nutritional support may be unintentionally deferred in critically ill patients due to fluid restriction , preparation for surgical / procedural interventions and/or concern for feeding intolerance . the resultant malnutrition in the setting of aki is associated with increased morbidity and mortality [ 23 , 24 ] . nutritional deficit , specifically protein debt , is linked to higher risk for infection , reduced wound healing and increased need for mechanical ventilation . provision of adequate nutritional support to critically ill patients is a major benefit of rst . the smallest of patients are at high risk for morbidity and mortality secondary to aki , yet there remains a dearth of clinical equipment and expertise available to provide adequate rst for these patients . technical adaptation is required for placement of pd catheters in elbw infants ; however , renal recovery following severe oligo - anuric aki is possible and can be followed by excellent growth , development and improvement in lung function . these infants should not be excluded from consideration for initiation of rst because of their small size .

critically ill neonates are at high risk for acute kidney injury ( aki ) . renal supportive therapy ( rst ) can be an important tool for supporting critically ill neonates with aki , particularly in cases of oliguria and fluid overload . there are few reports of rst for management of oligo - anuric aki in the extremely low - birth - weight infant weighing < 1000 g. we report successful provision of peritoneal dialysis ( pd ) to an 830-g neonate with oligo - anuric aki through adaptation of a standard pediatric acute pd catheter .

Background Case report Procedure Discussion Conclusion

PMC4935702

despite the fact that women s health issues is coming to the forefront of health priorities in many countries , yet , it was observed that women in developing world remain susceptible to poor health because of lack of services and lack of education and information about health issues ( 1 ) . in developing countries , complications of pregnancy and childbirth are the leading causes of death among women of reproductive age . women risk death and disability each time they become pregnant ; and face these risks much more often since they bear many more children than women in the developed world . who report that women s risk of dying from pregnancy related complications stood at 1 in 48 for all developing countries as compared to 1 in 1,800 for developed countries meaning the risk of dying from pregnancy related complications are highest in africa ( 2 ) . delivery in health facility by skilled birth attendant is recommended for all pregnant women as a strategy to reduce maternal and neonatal mortality ( 3 , 4 ) . delivery in health facility allow for proper management of labor , early detection of problem in both the mother and the fetus in the event of complications which are usually unpredictable it allows for prompt and effective response . we report findings from zamfara state in northwest nigeria , where an on - going health system strengthening project with technical and financial support from department for international development ( dfid ) /norwegian government in collaboration with the state government aimed at addressing challenges related to maternal and child health in all its ramification , this provide a unique opportunity to understand why women are averse to facility delivery even when facility based delivery is available at minimal cost . the study was conducted in sub - urban and rural settings in ( zamfara state ) northwest nigeria between may 2012 and mar 2013 . its population as at the 2006 national population and housing census was 3,259,846 ( 1,630,344 males and 1,629,502 females ) ( 5 ) . the public healthcare system is made up of primary , secondary and tertiary health facilities . there are more than 600 primary health centers , 18 general hospitals with 1 federal medical centre , in addition , 23 health facilities are privately owned . the local government areas ( lgas ) are responsible for the construction and running of village dispensaries , basic health clinics and primary health centers . the state ministry of health and health services management board are responsible for the secondary healthcare system . key informant interview ( kii ) and focus group discussion participants were selected purposively and saturation of information was used to decide on adequacy of the samples . ethical approval for this study was obtained from state operational research committee ( orac ) and informed verbal consent was obtained from all participants . the discussions involved five separate groups of women ( those who attended antenatal care ( anc ) , but delivered at home , those who delivered once in facility but fail to return on subsequent deliveries and those who neither had anc nor deliver in facility , mother - in - laws and men ( husbands ) who s wives opted for home / facility delivery for the most recent births . in addition , in - depth interviews were held with six health care providers ( nurses / chews ) and six traditional birth attendants ( tba ) . fgd categories in the communities characteristics of participants in focus group discussion and in depth - interview five different interview guide were used for the different sub - groups , trying to explore entrenched socio - cultural / religious barriers to facility delivery within and between categories of women , tba and health workers ( table 3 ) . the questions included issues affecting who decide when and why a woman should be taken to facility for delivery , what are the motivations for patronizing tba , and what are the experiences of those that used the facility for delivery . in addition we explored the health workers perspectives on why women are averse to facility delivery . main themes and sub - themes from fgds and kii data on barriers to delivery in health facilities the study teams consisted of the principal researcher , five female secondary school teachers with past experience in fgd one male undergraduate student of sociology of usmanu danfodio university , sokoto . the research teams participated in a two - days training ; the session included : study overview , ethical conduct of research , role play and pre - testing the study instruments . basic demographic data such as age parity and location of participants were collected from each study participant before the start of each focus group . all focus groups were held in hausa , the local language familiar to all research participants . the focus groups lasted about an hour each ; interviewers took extensive notes , in addition to tape recording and transcribing the interviews . the demographic data were processed using spss version 16 ( chicago , il , usa ) , and information obtained were transcribed verbatim . this textual data was compared and combined with information notes taken during the interview sessions . in addition , the content of the textual data was double checked with summaries and conclusions made at the end of the interview sessions in order to ensure that those notes were similar to what they answered during the interviews . basic description of data was done through open coding where textual data was decomposed into parts , marked and coded with the aid of nvivo software . then , the parts were compared based on the similarities and differences that combined to form a new category and subcategories . this was followed by linking the various categories , this is often referred to as axial coding , and the information of each category and it is subcategories was refined and determined in relation to its conditions , context , strategies and consequences . the study used a qualitative method ( phenomenology ) . key informant interview ( kii ) and focus group discussion participants were selected purposively and saturation of information was used to decide on adequacy of the samples . ethical approval for this study was obtained from state operational research committee ( orac ) and informed verbal consent was obtained from all participants . the discussions involved five separate groups of women ( those who attended antenatal care ( anc ) , but delivered at home , those who delivered once in facility but fail to return on subsequent deliveries and those who neither had anc nor deliver in facility , mother - in - laws and men ( husbands ) who s wives opted for home / facility delivery for the most recent births . in addition , in - depth interviews were held with six health care providers ( nurses / chews ) and six traditional birth attendants ( tba ) . fgd categories in the communities characteristics of participants in focus group discussion and in depth - interview five different interview guide were used for the different sub - groups , trying to explore entrenched socio - cultural / religious barriers to facility delivery within and between categories of women , tba and health workers ( table 3 ) . the questions included issues affecting who decide when and why a woman should be taken to facility for delivery , what are the motivations for patronizing tba , and what are the experiences of those that used the facility for delivery . in addition we explored the health workers perspectives on why women are averse to facility delivery . main themes and sub - themes from fgds and kii data on barriers to delivery in health facilities the study teams consisted of the principal researcher , five female secondary school teachers with past experience in fgd one male undergraduate student of sociology of usmanu danfodio university , sokoto . the research teams participated in a two - days training ; the session included : study overview , ethical conduct of research , role play and pre - testing the study instruments . basic demographic data such as age parity and location of participants were collected from each study participant before the start of each focus group . all focus groups were held in hausa , the local language familiar to all research participants . the focus groups lasted about an hour each ; interviewers took extensive notes , in addition to tape recording and transcribing the interviews . the demographic data were processed using spss version 16 ( chicago , il , usa ) , and information obtained were transcribed verbatim . this textual data was compared and combined with information notes taken during the interview sessions . in addition , the content of the textual data was double checked with summaries and conclusions made at the end of the interview sessions in order to ensure that those notes were similar to what they answered during the interviews . basic description of data was done through open coding where textual data was decomposed into parts , marked and coded with the aid of nvivo software . then , the parts were compared based on the similarities and differences that combined to form a new category and subcategories . this was followed by linking the various categories , this is often referred to as axial coding , and the information of each category and it is subcategories was refined and determined in relation to its conditions , context , strategies and consequences . the concept and idea of antenatal care ( anc ) enjoyed universal acceptability among the participants in all the communities studied , with low utilization of facilities for delivery . women who delivered outside health facilities relate their circumstances to the following factors , getting permission from husband / family , distance to a health facility or lack of transportation , cost of service fee for treatment , trust in service quality , belief that it may not be necessary or customary ( fig . reasons for not delivering in facilities in addition , cultural sensitivity , social support , availability and affordability of services , previous negative experiences with health facilities delivery , cultural perception of the role of tbas , attitude of health workers to clients , clients assessment of quality of care , ignorance/ low awareness of facility services , economic constraints , and relative autonomy of women / empowerment were among other findings . analyses of the data revealed two patterns in terms of the responses , the general and the specific . the general related to responses common to all the groups and the second related to themes gleaned from the specific responses of the different groups of participants as outlined above . the general related to issues that were found to commonly emerge from the narratives of all the participants in all categories , male and female relating to perceptions and practices on traditional and modern management of pregnancies and births within the local culture . the specific , on the other hand , related to analyses of narratives of participants in fgds with specific groups of women on their different experiences , supported by data from the kii interviews . most of the respondents were stoic about the possibility of other interventions which , at any rate , were in the realm of divine providence pertaining to their occurrence and resolution . a key indicator of such a perception was the very frequent use of the ( hausa ( local language ) word matsala to mean complications / problems , etc . ) . labor is now short , quick and uncomplicated and if you go to the hospital for delivery , people will think problems have emerged and you need assistance . allah is helping us it is a known fact that most facilities are located in the urban areas and less , in the rural areas . according to a young mother , hospital delivery was.very important ; that is why in the town when it is time you just pick your kit and go there to deliver but in the village we do nt do that unless there is a problem . in short , across all categories , male and female , the participants did not see facility delivery as a necessity , or even a priority , as obvious in such opinions expressed in fgds by anc - regular women during the fgd sessions : yes , attending for anc is important but you only go there to deliver if you become weak or can not deliver on your own without assistance one goes there only if a problem develops or when the labour takes much time typical indications about this possibility include the following statements attended from two women who regularly accessed anc services : .. i went for anc but no one told me to return for delivery .they only said that if you have any problem you go to the hospital but nobody will tell you that when it is time for delivery we should go to the hospital in contrast facility delivery clearly did not enjoy popularity . several misgivings or disincentives were expressed by almost all the participants , males and females , irrespective of age , location , specific previous experience with facilities , etc . the misgivings related to discomfort or dislike with specific procedures or bureaucracy at facilities , including : restricted or limited availability or accessibility to social support network , ( family members , relations , friends , and other significant others ) ; cumbersome procedures , such as requirements to pay for certain services ( such as tests ) . a female participant stated that sometimes they collect money , but it was not clear if such payment(s ) were official or as gratifications offered / demanded . however , a young female regular anc attendee added that if drugs are available , they give us and if not they ask us to go and buy . fixation / obsession with hospital cards ( kati , understood to include registration cards and files ) , found or viewed as modern irritants ; rigid working hours observed by facility staff , especially in the rural areas such that the staff were available only at specified times ( business hours ) and days ( no service on weekends ) ; rejection of or delays in attending to women showing up for delivery ( often due to complications ) but who had not been attending anc . such people were often berated and spoken to in harsh tones . a contributor narrated the experience of an acquaintance who took his ( acquaintance s ) wife , in a critical condition to a health facility . the wife needed immediate attention ( rai a hannun allah or who looked like she could die at any moment ) only for the health worker to berate the husband as a villager who only now is bringing a corpse to the facility , even as the patient could not indeed be helped at the location : as if the insult was not bad enough , they were .. referred us to the hospital at gusau . ultimately , the wife died for lack of attention . words used to describe such experiences or perceptions and attitudes translated into arrogance , impatience , lack of empathy , partiality ( tending to pay greater attention to elite women ) ; poor public relations , etc . rendered in hausa words and phrases conveying such negative attributions , such as cin zarafi , tozartarwa and wulakanchi ( an elderly husband related his wife s experience of reaching a facility apparently with a complication ( probably an obstructed labour ) only to be called .. a lazy villager . ironically , my wife delivered safely on her own even before the midwife got round to attend to her . my wife then asked the midwife who the villager was in that situation . since then my wife had vowed never to return to the hospital . she had even instructed me never to be taken to the facility for delivery if it ever became obvious that a delivery would end her life , she would rather die than accept that the medical treatment given with humiliation . another recounted a discouraging experience thus we rushed my friend s wife to the hospital only to be told that the doctor was at home . the condition of my friend s wife was visibly deteriorating and the nurses appeared helpless . my friend lost it , went wild , grabbed the doctor by the collar and threatened to kill him ( doctor ) should the wife in labour die ! it was only then that the doctor attended to the woman and she eventually delivered . that story travelled far these kinds of stories are unpleasant and do not inspire or encourage positive attitudes to hospitals and the doctors a woman who had indeed even given up on anc visits claimed to have so decided because a facility staff used to shout on people such that now i do nt go there . specific findings unique to groups for women who neither went for anc nor go to facilities to deliver , there was deep - seated preference for the home , and apprehension about , the facilities , virtually all , the major attractions of home delivery were , foremost , low costs and the privacy that the home environment offered . in particular , the home environment resonated with a desire not to be publicly exposed to non - relations during the birth process , in line with cultural practices in this predominantly muslim hausa and fulani majority environment , where reticence and stoicism were expected of persons undergoing physical and psychological pains / challenges . sensitivity of the home environment to entrenched cultural practices ( and , in particular , clear separation between males and females ) was thus a critical deciding factor . further , the home environment allowed the woman in labor to be attended to by tbas , as were the availability of extensive network of social support including husbands , mothers , relations , co - wives , etc . , who readily rendered assistance , relieve women in labor of responsibility for household chores , etc . such social support also allowed pregnant women to do things that were not allowed or permitted in hospitals ( e.g. access to foods and drinks ) . in short , the home environment was such an entrenched and very supportive comfort zone incomparable to the open ( public ) nature of facilities . for such women , modern health facilities offered no guarantees ( for safe deliveries ) anyway , as shown in the following narratives : after all , these were what we grew up to know and had been used for generations ! and : even health facilities can not prevent divine ordination in case of the outcome of complications . it needs be stated here that generational differences were found among women in this category : older women expressed greater confidence in traditional remedies , while younger women expressed doubts about the efficacy of traditional remedies calling them hade - hade ( or concoctions of unknown efficacy , given on trial - and - error basis ) . thus , for the younger women , except for other factors , facility deliveries would be preferable to home delivery . still , and not surprisingly , both the older and younger generation of the women were aware of the shortcomings of the home environment , including the lack of facilities and expertise to handle complications that may arise ( obstructed labor , excessive bleeding , retained placentas , etc . ) . while the older women hesitated to admit the inadequacy of the home environment to deal with such eventualities ( complications ) , the younger ones were more open to admitting the doubtful / unreliable efficacies and effectiveness of herbal concoctions , and other home remedies ( i.e. the hade - hade alluded to above ) . this category of women were clear about the fact of modern health facilities having advantages relating , especially , to monitoring the pregnancy process and offering effective management of birth complications , as well as the availability of modern reliable and effective drugs . additionally , as revealed by a participant at a rural location during an fgd session , an additional attraction going for facility delivery was the incentives given to patrons such as baby kits and mosquito nets . the gifts had become symbols of conformity to modern care that women now compete to acquire and show off , as indeed confirmed by a facility worker at a kii session . another woman given up on anc visit explained that she was put off because of a facility staff : she used to shout on people but now i do nt know because i do nt go there again . this category of women expressed a lot of the sentiments as well as re - echoed perceptions of facilities as expressed by participants of the other categories treated above . however , their most frequently mentioned disincentives for going to facilities to deliver were : ( a ) costs associated with transportation fare , especially for those who could only attend facility outside their villages / localities . a male household head and participant at an fgd session in a rural location , was emphatic that the problem revolved around talauchii , ( translated to condition material poverty ) make no mistake about the bush on this facility delivery thing : our problem is poverty ; our daily struggles relate to feeding our families . you are thinking of how to feed the family , buy a ram to slaughter to celebrate births , you hardly boast of n5.00 and you struggle with transportation to reach the hospital with a woman in labor only to be confronted with a list of things to purchase . god save you if you the wife now needs surgery which can costs thousands of naira the simple truth is government has to help us ( a ) costs associated with transportation fare , especially for those who could only attend facility outside their villages / localities . a male household head and participant at an fgd session in a rural location , was emphatic that the problem revolved around talauchii , ( translated to condition material poverty ) make no mistake about the bush on this facility delivery thing : our problem is poverty ; our daily struggles relate to feeding our families . you are thinking of how to feed the family , buy a ram to slaughter to celebrate births , you hardly boast of n5.00 and you struggle with transportation to reach the hospital with a woman in labor only to be confronted with a list of things to purchase . god save you if you the wife now needs surgery which can costs thousands of naira the simple truth is government has to help us reflecting such resignation to fate / stoic philosophy a tba expressed during a kii that : .. a pregnant should indeed be taken care of and people do try within the limits of allah s provision for them , but you know we are villagers ; some people try but some just ca nt afford to . similarly a woman participant of this category complained that they also collect money for blood tests and you must pay for that or they will not attend to you ( b ) shyness and reticence , and deep dislike / distaste for being touched by males other than their husbands ; there are others disallowed by their husbands , from going to the clinic because such husbands could not contemplate their wives being attended to by strange males . ( c ) distaste for blood transfusion and being forced to accept vaccination ; fear of surgical interventions ( including any procedure that involved cutting and stitching parts of the body , episiotomy , etc . ) . there was a wide belief that all too often facility workers were quick in deciding to go for surgical interventions , even when the need for same was not obvious to husbands / relations accompanying pregnant women to the hospital . several participants recalled situations where pregnant women delivered babies on their own as preparations were being made to admit them into labor rooms or theatres for caesarian operations ( cs ) at facilities . another recounted a discouraging experience thus we rushed my friend s wife to the hospital only to be told that the doctor was at home . the condition of my friend s wife was visibly deteriorating and the nurses appeared helpless . my friend lost it , went wild , grabbed the doctor by the collar and threatened to kill him ( doctor ) should the wife in labor die ! it was only then that the doctor attended to the woman and she eventually delivered . that story travelled far these kinds of stories are unpleasant and do not inspire or encourage positive attitudes to hospitals and the doctors a woman who had indeed even given up on anc visits claimed to have so decided because a facility staff used to shout on people such that now i do nt go there . the traditional birth attendants ( tba ) that participated in this study were mostly elderly women , and often conduct delivery alone . most tbas inherit the role from their mothers , they are normally called only after delivery or when a complication has set in , and may not be around at critical times when danger signs need to be identified . tbas cut the cord and bury the placenta , bath the baby for seven days and assist women to take a hot bath for forty days after delivery . a tba expressed during a kii that : a pregnant should indeed be taken care of and people do try within the limits of god s provision for them , but you know we are villagers ; some people try but some just ca nt afford to similarly a woman participant of this category complained that they also collect money for blood tests and you must pay for that or they will not attend to you shyness and reticence , and deep dislike / distaste for being touched by males other than their husbands ; there are others disallowed by their husbands , from going to the clinic because such husbands could not contemplate their wives being attended to by strange males . there was a wide spread belief that all too often facility workers were quick in deciding to go for surgical interventions , even when the need for same was not obvious to husbands / relations accompanying pregnant women to the hospital . several participants recalled situations where pregnant women delivered babies on their own as preparations were being made to admit them into labor rooms or theatres for caesarian operations ( cs ) at facilities . most of the respondents were stoic about the possibility of other interventions which , at any rate , were in the realm of divine providence pertaining to their occurrence and resolution . a key indicator of such a perception was the very frequent use of the ( hausa ( local language ) word matsala to mean complications / problems , etc . ) . labor is now short , quick and uncomplicated and if you go to the hospital for delivery , people will think problems have emerged and you need assistance . allah is helping us it is a known fact that most facilities are located in the urban areas and less , in the rural areas . according to a young mother , hospital delivery was.very important ; that is why in the town when it is time you just pick your kit and go there to deliver but in the village we do nt do that unless there is a problem . in short , across all categories , male and female , the participants did not see facility delivery as a necessity , or even a priority , as obvious in such opinions expressed in fgds by anc - regular women during the fgd sessions : yes , attending for anc is important but you only go there to deliver if you become weak or can not deliver on your own without assistance one goes there only if a problem develops or when the labour takes much time typical indications about this possibility include the following statements attended from two women who regularly accessed anc services : .. i went for anc but no one told me to return for delivery .they only said that if you have any problem you go to the hospital but nobody will tell you that when it is time for delivery we should go to the hospital in contrast facility delivery clearly did not enjoy popularity . several misgivings or disincentives were expressed by almost all the participants , males and females , irrespective of age , location , specific previous experience with facilities , etc . the misgivings related to discomfort or dislike with specific procedures or bureaucracy at facilities , including : restricted or limited availability or accessibility to social support network , ( family members , relations , friends , and other significant others ) ; cumbersome procedures , such as requirements to pay for certain services ( such as tests ) . a female participant stated that sometimes they collect money , but it was not clear if such payment(s ) were official or as gratifications offered / demanded . however , a young female regular anc attendee added that if drugs are available , they give us and if not they ask us to go and buy . fixation / obsession with hospital cards ( kati , understood to include registration cards and files ) , found or viewed as modern irritants ; rigid working hours observed by facility staff , especially in the rural areas such that the staff were available only at specified times ( business hours ) and days ( no service on weekends ) ; rejection of or delays in attending to women showing up for delivery ( often due to complications ) but who had not been attending anc . a contributor narrated the experience of an acquaintance who took his ( acquaintance s ) wife , in a critical condition to a health facility . the wife needed immediate attention ( rai a hannun allah or who looked like she could die at any moment ) only for the health worker to berate the husband as a villager who only now is bringing a corpse to the facility , even as the patient could not indeed be helped at the location : as if the insult was not bad enough , they were .. referred us to the hospital at gusau . ultimately , the wife died for lack of attention . do you think we would ever take our wives to the hospital ? negative attitudes of facility workers , a rampant complaint . words used to describe such experiences or perceptions and attitudes translated into arrogance , impatience , lack of empathy , partiality ( tending to pay greater attention to elite women ) ; poor public relations , etc . rendered in hausa words and phrases conveying such negative attributions , such as cin zarafi , tozartarwa and wulakanchi ( an elderly husband related his wife s experience of reaching a facility apparently with a complication ( probably an obstructed labour ) only to be called .. a lazy villager . ironically , my wife delivered safely on her own even before the midwife got round to attend to her . my wife then asked the midwife who the villager was in that situation . since then my wife had vowed never to return to the hospital . she had even instructed me never to be taken to the facility for delivery if it ever became obvious that a delivery would end her life , she would rather die than accept that the medical treatment given with humiliation . another recounted a discouraging experience thus we rushed my friend s wife to the hospital only to be told that the doctor was at home . the condition of my friend s wife was visibly deteriorating and the nurses appeared helpless . my friend lost it , went wild , grabbed the doctor by the collar and threatened to kill him ( doctor ) should the wife in labour die ! it was only then that the doctor attended to the woman and she eventually delivered . that story travelled far these kinds of stories are unpleasant and do not inspire or encourage positive attitudes to hospitals and the doctors a woman who had indeed even given up on anc visits claimed to have so decided because a facility staff used to shout on people such that now i do nt go there . for women who neither went for anc nor go to facilities to deliver , there was deep - seated preference for the home , and apprehension about , the facilities , virtually all , the major attractions of home delivery were , foremost , low costs and the privacy that the home environment offered . in particular , the home environment resonated with a desire not to be publicly exposed to non - relations during the birth process , in line with cultural practices in this predominantly muslim hausa and fulani majority environment , where reticence and stoicism were expected of persons undergoing physical and psychological pains / challenges . sensitivity of the home environment to entrenched cultural practices ( and , in particular , clear separation between males and females ) was thus a critical deciding factor . further , the home environment allowed the woman in labor to be attended to by tbas , as were the availability of extensive network of social support including husbands , mothers , relations , co - wives , etc . , who readily rendered assistance , relieve women in labor of responsibility for household chores , etc . such social support also allowed pregnant women to do things that were not allowed or permitted in hospitals ( e.g. access to foods and drinks ) . in short , the home environment was such an entrenched and very supportive comfort zone incomparable to the open ( public ) nature of facilities . for such women , modern health facilities offered no guarantees ( for safe deliveries ) anyway , as shown in the following narratives : after all , these were what we grew up to know and had been used for generations ! and : even health facilities can not prevent divine ordination in case of the outcome of complications . it needs be stated here that generational differences were found among women in this category : older women expressed greater confidence in traditional remedies , while younger women expressed doubts about the efficacy of traditional remedies calling them hade - hade ( or concoctions of unknown efficacy , given on trial - and - error basis ) . thus , for the younger women , except for other factors , facility deliveries would be preferable to home delivery . still , and not surprisingly , both the older and younger generation of the women were aware of the shortcomings of the home environment , including the lack of facilities and expertise to handle complications that may arise ( obstructed labor , excessive bleeding , retained placentas , etc . ) . while the older women hesitated to admit the inadequacy of the home environment to deal with such eventualities ( complications ) , the younger ones were more open to admitting the doubtful / unreliable efficacies and effectiveness of herbal concoctions , and other home remedies ( i.e. the hade - hade alluded to above ) . this category of women were clear about the fact of modern health facilities having advantages relating , especially , to monitoring the pregnancy process and offering effective management of birth complications , as well as the availability of modern reliable and effective drugs . additionally , as revealed by a participant at a rural location during an fgd session , an additional attraction going for facility delivery was the incentives given to patrons such as baby kits and mosquito nets . the gifts had become symbols of conformity to modern care that women now compete to acquire and show off , as indeed confirmed by a facility worker at a kii session . another woman given up on anc visit explained that she was put off because of a facility staff : she used to shout on people but this category of women expressed a lot of the sentiments as well as re - echoed perceptions of facilities as expressed by participants of the other categories treated above . however , their most frequently mentioned disincentives for going to facilities to deliver were : ( a ) costs associated with transportation fare , especially for those who could only attend facility outside their villages / localities . a male household head and participant at an fgd session in a rural location , was emphatic that the problem revolved around talauchii , ( translated to condition material poverty ) make no mistake about the bush on this facility delivery thing : our problem is poverty ; our daily struggles relate to feeding our families . you are thinking of how to feed the family , buy a ram to slaughter to celebrate births , you hardly boast of n5.00 and you struggle with transportation to reach the hospital with a woman in labor only to be confronted with a list of things to purchase . god save you if you the wife now needs surgery which can costs thousands of naira the simple truth is government has to help us ( a ) costs associated with transportation fare , especially for those who could only attend facility outside their villages / localities . a male household head and participant at an fgd session in a rural location , was emphatic that the problem revolved around talauchii , ( translated to condition material poverty ) make no mistake about the bush on this facility delivery thing : our problem is poverty ; our daily struggles relate to feeding our families . you are thinking of how to feed the family , buy a ram to slaughter to celebrate births , you hardly boast of n5.00 and you struggle with transportation to reach the hospital with a woman in labor only to be confronted with a list of things to purchase . god save you if you the wife now needs surgery which can costs thousands of naira the simple truth is government has to help us reflecting such resignation to fate / stoic philosophy a tba expressed during a kii that : .. a pregnant should indeed be taken care of and people do try within the limits of allah s provision for them , but you know we are villagers ; some people try but some just ca nt afford to . similarly a woman participant of this category complained that they also collect money for blood tests and you must pay for that or they will not attend to you ( b ) shyness and reticence , and deep dislike / distaste for being touched by males other than their husbands ; there are others disallowed by their husbands , from going to the clinic because such husbands could not contemplate their wives being attended to by strange males . ( c ) distaste for blood transfusion and being forced to accept vaccination ; fear of surgical interventions ( including any procedure that involved cutting and stitching parts of the body , episiotomy , etc . ) . there was a wide belief that all too often facility workers were quick in deciding to go for surgical interventions , even when the need for same was not obvious to husbands / relations accompanying pregnant women to the hospital . several participants recalled situations where pregnant women delivered babies on their own as preparations were being made to admit them into labor rooms or theatres for caesarian operations ( cs ) at facilities . another recounted a discouraging experience thus we rushed my friend s wife to the hospital only to be told that the doctor was at home . the condition of my friend s wife was visibly deteriorating and the nurses appeared helpless . my friend lost it , went wild , grabbed the doctor by the collar and threatened to kill him ( doctor ) should the wife in labor die ! it was only then that the doctor attended to the woman and she eventually delivered . that story travelled far these kinds of stories are unpleasant and do not inspire or encourage positive attitudes to hospitals and the doctors a woman who had indeed even given up on anc visits claimed to have so decided because a facility staff used to shout on people such that now i do nt go there . the traditional birth attendants ( tba ) that participated in this study were mostly elderly women , and often conduct delivery alone . most tbas inherit the role from their mothers , they are normally called only after delivery or when a complication has set in , and may not be around at critical times when danger signs need to be identified . tbas cut the cord and bury the placenta , bath the baby for seven days and assist women to take a hot bath for forty days after delivery . a tba expressed during a kii that : a pregnant should indeed be taken care of and people do try within the limits of god s provision for them , but you know we are villagers ; some people try but some just ca nt afford to similarly a woman participant of this category complained that they also collect money for blood tests and you must pay for that or they will not attend to you shyness and reticence , and deep dislike / distaste for being touched by males other than their husbands ; there are others disallowed by their husbands , from going to the clinic because such husbands could not contemplate their wives being attended to by strange males . there was a wide spread belief that all too often facility workers were quick in deciding to go for surgical interventions , even when the need for same was not obvious to husbands / relations accompanying pregnant women to the hospital . several participants recalled situations where pregnant women delivered babies on their own as preparations were being made to admit them into labor rooms or theatres for caesarian operations ( cs ) at facilities . this study shows that there is good acceptance of antenatal care and it will result in safe delivery . the major and common reasons across board for preference for home delivery include poverty , affordability , facility delivery is alien , and unattractive to them and inadequate / lack of empathy from facility staff when compare with empathy and support from relation at home . the home preference is largely related to cost , with hospital deliveries known to be very expensive . one man mentioned the sum of naira 2,000 ( equivalent to twelve dollars ) for a routine hospital delivery . tbas are usually paid in kind , with anything within your reach , even farm produce . a narrative by one male regard to tbas was : we were born through their services , and we are around today. similar phenomena are highlighted by kruk et al finding in rural tanzania study ( 9).but in contrast to orisaremi s finding among tarok women in north - central nigeria , where bravery and audacity are the main factors that discourage most women from facility delivery ( 10 ) . apart from user fee being unaffordable to many , the payment procedure is cumbersome and no credit facility , instalment payment or exchange of goods for service as practiced by traditional birth attendants , which provide succour to many families . in shah s study in karachi , india , the two most common reasons for home deliveries are family tradition and lack of affordability / poor socio - economic condition of the family ( 11 ) . lack of privacy and exposure to non - relatives and men and dislike / distaste for being touched by male other than their husband which is borne out of cultural and / or religious belief are another major factors that drive women away from facility delivery in this study . since there is no guarantee that they will meet a female staff at time of labour . some attitudes of the hospital staff and hospital practices such as shouting on patients , arrogance , impatience , humiliation , lack of empathy and abuse , tapping patients in labour , all these discourage women from facility delivery . many women can not bear it and that is one of the reasons why they use facility as last resort when they have no other option . this study also reveal perception about facility delivery which is seen to be as necessary if complications emerged , a last resort , an aide in the management of difficult births or those that come with complications . most of the respondents are stoic about the possibility of other interventions which , at any rate , are in the realm of divine providence pertaining to their occurrence and resolution . among the less common reasons for aversion to facility delivery in this study is fear of surgical intervention . this is the most common reason among guatemala women for not going to facility for delivery even in the case of life threatening complications ( 12 ) . the caesarean section is often thought to be unnecessary and may have effect on future fertility . there is a generational difference in perception and attitude towards facility delivery between younger and older generation of women . most of the women are aware of the possible drawbacks of home delivery such as lack of facilities ( equipment ) , expertise to recognize early complication signs and management of complication . based on these findings it is recommended that facility services should be subsidized if not free , girls / women should be educated and empowered , health care providers should have seminars on attitudinal change so as to cultivate positive attitudes , traditional birth attendants should be officially engaged in encouraging women to embrace facility delivery and provision of incentives such as mosquito nets and baby kits to encourage facility delivery . the home preference is largely related to cost , with hospital deliveries known to be very expensive . one man mentioned the sum of naira 2,000 ( equivalent to twelve dollars ) for a routine hospital delivery . tbas are usually paid in kind , with anything within your reach , even farm produce . a narrative by one male regard to tbas was : we were born through their services , and we are around today. similar phenomena are highlighted by kruk et al finding in rural tanzania study ( 9).but in contrast to orisaremi s finding among tarok women in north - central nigeria , where bravery and audacity are the main factors that discourage most women from facility delivery ( 10 ) . apart from user fee being unaffordable to many , the payment procedure is cumbersome and no credit facility , instalment payment or exchange of goods for service as practiced by traditional birth attendants , which provide succour to many families . in shah s study in karachi , india , the two most common reasons for home deliveries are family tradition and lack of affordability / poor socio - economic condition of the family ( 11 ) . lack of privacy and exposure to non - relatives and men and dislike / distaste for being touched by male other than their husband which is borne out of cultural and / or religious belief are another major factors that drive women away from facility delivery in this study . since there is no guarantee that they will meet a female staff at time of labour . some attitudes of the hospital staff and hospital practices such as shouting on patients , arrogance , impatience , humiliation , lack of empathy and abuse , tapping patients in labour , all these discourage women from facility delivery . many women can not bear it and that is one of the reasons why they use facility as last resort when they have no other option . this study also reveal perception about facility delivery which is seen to be as necessary if complications emerged , a last resort , an aide in the management of difficult births or those that come with complications . most of the respondents are stoic about the possibility of other interventions which , at any rate , are in the realm of divine providence pertaining to their occurrence and resolution . among the less common reasons for aversion to facility delivery in this study this is the most common reason among guatemala women for not going to facility for delivery even in the case of life threatening complications ( 12 ) . the caesarean section is often thought to be unnecessary and may have effect on future fertility . there is a generational difference in perception and attitude towards facility delivery between younger and older generation of women . most of the women are aware of the possible drawbacks of home delivery such as lack of facilities ( equipment ) , expertise to recognize early complication signs and management of complication . based on these findings it is recommended that facility services should be subsidized if not free , girls / women should be educated and empowered , health care providers should have seminars on attitudinal change so as to cultivate positive attitudes , traditional birth attendants should be officially engaged in encouraging women to embrace facility delivery and provision of incentives such as mosquito nets and baby kits to encourage facility delivery . the common reasons for aversion to facility delivery include poverty , cost of service , non - permission from husband , alien and negative attitude of staff . other reasons include its unnecessary , lack of privacy and exposure to non - relatives and men and dislike / distaste for being touched by male other than their husband and lack of empathy . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .

background : in many sub - saharan african countries the rate of antenatal care ( anc ) has been increased but skilled birth attendance rate is still low . the objective of this study was to evaluate the reasons why women prefer home delivery when facility based delivery is available at minimal cost.methods:this study was conducted in northwest nigeria using a qualitative method ( phenomenology ) among five categories of women in april may 2013 . this study investigated different categories of women ( those that never attend anc nor deliver in the facility , those that attend anc but delivered at home and those that delivered once in the facility but fail to return in subsequent deliveries , the in - laws and facilities staff).results : through focus group discussions and in - depth interviews several reasons why women are averse to hospital deliveries were identified . women reported ignorance , abuse , illiteracy , and poverty , and low esteem , poor attitude of health workers , few working hours and some integrated health services like preventing mother to child transmission of hiv testing as deterrents , while cheap and accessible services were reasons for preference to traditional birth attendants.conclusions:the findings highlighted important entrenched barriers to facility deliveries among women , which is basically socio - cultural and economic . therefore emphasis must be given to health education program to ensure comprehensive and target specific messages that will address individual needs of the groups .

Introduction Materials and Methods Study Design and Sampling Data analyses Results General response Perceptions of the Home and Facility Environments Among Women who Neither Accessed ANC nor Health Facilities for Delivery Women who delivered once but fail to return in subsequent deliveries FGD with women who attend ANC but delivered at home Traditional Birth Attendants Discussion Mother/Father In-laws Conclusion Ethical considerations

PMC4033635

cellular genomes are constantly damaged by endogenous agents , such as oxygen radicals formed during aerobic respiration . this oxidative dna damage is proposed to be a major contributor to carcinogenesis and aging . one of the most common oxidative dna lesions generated within cells is 8-oxo-7,8-dihydro-2-deoxyguanosine ( 8-oxodg ) . although structural studies indicate that 8-oxodg induces only minor distortions to the helical structure of dna that are localized to the modified base , 8-oxodg is highly mutagenic due to its dual coding potential . during dna replication , 8-oxodg adopts either an anti or syn conformation within a polymerase active site . while in the syn conformation , a templating 8-oxodg lesion utilizes the hoogsteen edge of the damaged base to preferentially form base pairs with incoming datp . if left unrepaired , these 8-oxodg : da mispairs will lead to g t transversion mutations , which have been linked to cancer induction . although 8-oxodg does not completely block dna synthesis catalyzed by replicative dna polymerases , this lesion does induce the pausing of dna polymerases alpha ( pol ) , delta ( pol ) , and epsilon ( pol ) . such a stalling event may provide an opportunity for a cell to switch to a dna polymerase that is specialized for the bypass of dna lesions , a process known as translesion dna synthesis ( tls ) . notably , humans encode four y - family enzymes : dna polymerases eta ( hpol ) , iota ( hpol ) , kappa ( hpol ) , and rev1 ( hrev1 ) . these four low - fidelity dna polymerases are characterized by a lack of proofreading 3 5 exonuclease activity , low processivity , and a relatively spacious , solvent - exposed active site . importantly , the human y - family enzymes demonstrate a large degree of functional divergence due to the fact that during tls , the nucleotide incorporation fidelity and efficiency of each y - family polymerase is lesion specific . therefore , it is likely that each y - family polymerase has evolved to catalyze tls of a specific set of lesions . investigation of the lesion bypass efficiencies and fidelities of the y - family polymerases may help illuminate the in vivo roles of these polymerases for the tls of specific lesions . to this end , earlier reports have examined the 8-oxodg bypass abilities of hpol , hpol , hpol , and hrev1 separately by using various enzymatic assays . however , these studies have utilized different reaction conditions and dna substrates to individually investigate each enzyme , complicating direct comparisons between the activities of all four human y - family enzymes . furthermore , the kinetic methods previously utilized were unable to accurately measure deletion or insertion events induced by tls . therefore , we sought to systematically compare the 8-oxodg bypass efficiency of each y - family enzyme under the same reaction conditions and to determine the type and frequency of errors induced by tls of 8-oxodg through a high - throughput short oligonucleotide sequencing assay ( ht - sosa ) that was recently developed in our laboratory . the ht - sosa method is advantageous for a comprehensive analysis of replication errors induced by tls of a damaged site as this method ( 1 ) provides a complete mutagenic profile of dna damage - induced mutations by accounting for all mutagenic events , such as base deletions , insertions , and substitutions ; ( 2 ) supplies sequencing information for incorporation events opposite , upstream , and downstream from the lesion site ; and ( 3 ) allows for analysis of multibase mutations from a single full - length product . here , we use a combination of primer extension assays and ht - sosa to investigate for the first time the efficiency and accuracy of 8-oxodg bypass catalyzed by the human y - family dna polymerases both alone and in combination . optikinase was purchased from usb corporation , dntps were purchased from ge healthcare , and [ -p]atp was purchased from perkinelmer . human pol with a c - terminal his6 tag , human pol with an n - terminal his6 tag , and human rev1 with an n - terminal his6 tag were expressed in escherichia coli and purified as previously described . human pol with an n - terminal gst tag was expressed in escherichia coli and purified , and the n - terminal gst tag was subsequently removed by proteolytic cleavage with tobacco etch viral protease as previously described . the synthetic 40-mer dna template containing 8-oxodg ( 40-mer-8oxodg ) was purchased from midland certified reagent company ( table 1 ) . all other synthetic dna oligomers were purchased from integrated dna technologies ( table 1 and table s1 , supporting information ) . all dna oligomers were gel purified by denaturing polyacrylamide gel electrophoresis ( page , 10% polyacrylamide , 8 m urea ) . primers were 5-[p]-radiolabeled by incubating each primer with optikinase and [ -p]atp for 3 h at 37 c . dna substrates were generated by annealing a 5-[p]-radiolabeled primer with either the control 40-mer template or damaged 40-mer-8oxodg template at a molar ratio of 1.00:1.15 . annealing solutions were heat denatured by incubation at 75 c for 5 min , followed by slow cooling to 25 c over several hours . all kinetic and ht - sosa reactions were performed in optimized reaction buffer r ( 50 mm hepes , ph 7.5 at 37 c , 50 mm nacl , 5 mm mgcl2 , 0.1 mm edta , 5 mm dtt , 10% glycerol , and 0.1 mg / ml bsa ) at 37 c . a preincubated solution containing either a single polymerase ( 100 nm ) or all four human y - family polymerases ( 25 nm each ) and 100 nm of a 5-[p]-radiolabeled dna substrate ( 17-mer/40-mer or 17-mer/40-mer-8oxodg ) was rapidly mixed with a solution of all four dntps ( 200 m each ) . the reaction mixtures were quenched at various times by the addition of 0.37 m edta . a rapid chemical quench flow apparatus ( kintek ) was used for kinetic experiments with reaction times ranging from milliseconds to several minutes . the reaction products were resolved by using denaturing page and quantified by using a typhoon trio ( ge healthcare ) . analysis of the running start assays was performed as previously described by calculating the relative lesion bypass efficiencies ( 8-oxodg bypass% ) of each polymerase as a function of reaction time . for each time point t , 8-oxodg bypass% was calculated as the ratio of the bypass events to the encounter events ( eq 1):1where the total 8-oxodg bypass events ( b ) was the sum of the concentrations of all products with sizes greater than or equal to the 22-mer , and the total 8-oxodg encounter events ( e ) was the summation of the 21-mer product concentration and the total 8-oxodg bypass events ( b ) . the 8-oxodg bypass% as calculated by eq 1 was then plotted as a function of reaction time . to compare the 8-oxodg bypass efficiency of each polymerase , the t50 was defined as the time required for each y - family polymerase to bypass 50% of the total 8-oxodg lesions encountered . this t50 value was calculated from the 8-oxodg bypass% plots assuming a constant rate of lesion bypass between the time point immediately before and the time point immediately after the bypass of 50% of the lesion sites encountered . a preincubated solution of 100 nm or 400 nm of hrev1 and 100 nm of a 5-[p]-radiolabeled dna substrate ( 21-mer/40-mer or 21-mer/40-mer-8oxodg ) was rapidly mixed with a solution containing all four dntps ( 200 m each ) . where indicated , hpol ( 300 nm ) was added to the reactions 30 s after the addition of the dntp solution . lesion bypass products were generated by mixing either the radiolabeled 13-mer/40-mer substrate or the 13-mer/40-mer-8oxodg substrate ( 100 nm ) into a solution containing a single enzyme ( 100 nm ) or all four enzymes ( 25 nm each ) . this solution was incubated briefly and subsequently mixed with a solution of all four dntps ( 200 m each ) . the reactions containing only hpol or only hpol were incubated at 37 c for 1 h , and the reactions containing only hpol or all 4 human y - family polymerases were incubated at 37 c for 4 h. to produce lesion bypass products with the combination of hrev1 and hpol , either the 16-mer/40-mer or 16-mer/40-mer-8oxodg dna substrate ( 100 nm ) was briefly preincubated with hrev1 ( 100 nm ) and subsequently mixed with a solution containing all four dntps ( 200 m each ) . after 10 or 60 s for the control or damaged dna substrate reactions , respectively , hpol ( 300 nm ) was added , and the reactions were incubated for an additional 1 h at 37 c . after generation of the lesion bypass products , the full - length , newly synthesized dna strands were effectively separated from the template strands by using denaturing page , as the 13-mer and 16-mer primers annealed 5 bases upstream of the 3-end of the 40-mer and 40-mer-8oxodg templates ( scheme 1 ) . next - generation sequencing libraries were generated by first amplifying each purified , single - stranded dna lesion bypass product by using 1 of 10 unique barcoded primers ( table s1 , supporting information ) and a common reverse primer . the pcr products were then gel purified , and the remaining adapter sequences required for next - generation sequencing were added in a second round of pcr by using illumina pcr primers 1 and 2 ( table s1 , supporting information ) . the completed sequencing libraries were then gel purified and mixed in equal molar ratios . the sequencing libraries generated from the lesion bypass products were then mixed with an equal molar amount of bacteriophage x sequencing libraries to increase sequence diversity . finally , the sequencing library solution was sequenced by using a hiseq 2000 dna sequencer ( illumina ) . to compare the ability of each human y - family dna polymerase to bypass 8-oxodg under identical in vitro conditions , we carried out running start primer extension assays . to this end , dna substrates were generated by annealing a 5-[p]-radiolabeled 17-mer primer to either the undamaged 40-mer template or the damaged 40-mer-8oxodg template , which contained a site - specifically placed 8-oxodg lesion ( table 1 and figure 1k ) . these dna substrates were then extended by the action of a y - family dna polymerase . the 8-oxodg lesion did not induce any noticeable stalling of hpol when compared to the undamaged control ( figure 1a and b ) . thus , the efficiency of hpol nucleotide incorporation was not inhibited by the lesion during the bypass or extension step of 8-oxodg tls . in contrast , hpol paused during incorporations both opposite 8-oxodg and during the following extension step as indicated by the transient accumulation of the 22-mer and 23-mer products ( figure 1c and d ) . this result is consistent with previous steady - state kinetic studies indicating that the efficiency of hpol nucleotide incorporation is reduced opposite this lesion and that during the extension step of tls , hpol more efficiently extends the 8-oxodg : da mismatch compared to the correct 8-oxodg : dc base pair . the 8-oxodg lesion significantly blocked nucleotide incorporation by hpol both opposite the lesion and for the extension step as indicated by the accumulation of the 21-mer and 22-mer products , respectively ( figure 1e and f ) . these two consecutive pause sites were also observed with the control substrate but to a lesser extent , as hpol is known to preferentially misincorporate dg opposite template dt and inefficiently extend the mismatch . this combination of events reduces hpol activity opposite template dts , a phenomenon known as the t stop . thus , the relative efficiency of nucleotide incorporation catalyzed by hpol opposite 8-oxodg and during the extension step of tls is predicted to be sequence - dependent . polymerase hrev1 failed to extend past the lesion site within 20 h ( figure 1h ) . this failure to produce full - length dna products was expected as hrev1 preferentially functions as a dctp transferase by predominantly incorporating dctp opposite dna lesions or undamaged bases . nevertheless , the running start assay indicated that the 8-oxodg lesion reduced the activity of this polymerase as hrev1 extended the control dna substrate to a 23-mer but was only capable of extending the damaged dna substrate to a 21-mer product ( figure 1 g and h ) . running start assays for the human y - family dna polymerases on undamaged and damaged dna templates . a preincubated solution containing 100 nm of 5-[p]-radiolabeled ( a , c , e , g , and i ) 17-mer/40-mer or ( b , d , f , h , and j ) 17-mer/40-mer-8oxodg and either 100 nm of the indicated dna polymerase or 25 nm of each y - family polymerase was rapidly mixed with a solution containing all 4 dntps ( 200 m each ) . the reaction mixtures were quenched at the indicated times with 0.37 m edta and resolved by using denaturing page . the sizes of important products are indicated , and the 22nd position is denoted with an asterisk ( * ) to indicate an incorporation opposite the 8-oxodg lesion site . the position of the 8-oxodg lesion within the template strand is indicated by a y. to determine the effect of 8-oxodg bypass in the presence of all four human y - family dna polymerases in vitro , a running start assay was performed with an equal molar concentration of all four human y - family enzymes ( figure 1i and j ) . the time required for all of the human y - family enzymes to bypass the 8-oxodg lesion was similar to that of hpol alone , suggesting that in the absence of any auxiliary factors , hpol dominates the replication of both undamaged and damaged dna . furthermore , as with hpol alone , no strong pause sites were observed opposite the lesion ( figure 1j ) . interestingly , the only strong pause site observed in the presence of all four y - family enzymes was an accumulation of the 18-mer product . the only y - family enzyme that produced this strong 18-mer product was hrev1 ( figure 1 g and h ) , suggesting that the accumulation of the 18-mer was due to a single extension by hrev1 , followed by sequestration of this 18-mer product by bound hrev1 . running start assays performed with the combination of hpol , hpol , and hpol lack this 18-mer product ( figure s1 , supporting information ) , indicating that the accumulation of the 18-mer product in the presence of all four enzymes is indeed due to hrev1 . we concluded that when the human y - family polymerases were tested in combination , hpol catalyzed the majority of the nucleotide incorporation events . to define the relative lesion bypass efficiency of each y - family polymerase by using established methods , the percent of lesions bypassed by each polymerase with respect to the total number of lesions encountered ( 8-oxodg bypass% ) within the autoradiograms shown in figure 1 was plotted as a function of reaction time ( figure s2 , supporting information ) . to compare the relative lesion bypass efficiency of each enzyme , the time required for each polymerase to bypass 50% of the 8-oxodg sites encountered ( t50 ) relative to the replication of 50% of the corresponding dg sites ( t50 ) this qualitative analysis confirmed that the activity of hpol was not reduced by the 8-oxodg lesion . in contrast , the lesion reduced the nucleotide incorporation rate of hpol and hpol by 2.2- and 4.5-fold , respectively . the calculated t50 values also indicated that hpol bypassed the 8-oxodg lesion 186-fold and 1160-fold faster than hpol and hpol , respectively . consistently , the efficiency of 8-oxodg bypass by all four human y - family enzymes combined was nearly identical to that of hpol alone . taken together , these data indicate that in the absence of auxiliary factors , hpol bypassed 8-oxodg more efficiently than either hpol or hpol. calculated as the time required to bypass 50% of the 8-oxodg sites . determined by using the 21-mer/40-mer and 21-mer/40-mer-8oxodg substrates . because hrev1 failed to extend the 17-mer primer past the lesion site within 20 h ( figure 1 h ) , we chose to evaluate the ability of hrev1 to bypass the 8-oxodg lesion by using a standing start primer extension assay . to this end , an undamaged 21-mer/40-mer or damaged 21-mer/40-mer-8oxodg dna substrate ( figure 2a ) was extended by the action of hrev1 such that the first nucleotide incorporation by hrev1 was opposite either undamaged dg or the 8-oxodg lesion site ( figure 2b and c ) . interestingly , the 8-oxodg lesion reduced the rate of nucleotide incorporation catalyzed by hrev1 opposite the lesion and eliminated the ability of hrev1 to extend the lesion bypass product as indicated by the lack of 23-mer product formation in the presence of 8-oxodg ( figure 2c ) . the plot of dg and 8-oxodg bypass ( figure 2d ) revealed that 15% of the 8-oxodg lesions were not bypassed within 30 s , compared to only 6% of the undamaged dg sites that were not bypassed within the same time . this result suggests that approximately 9% of the hrev1 that encountered 8-oxodg formed nonproductive complexes due to interactions with the lesion . analyses of the standing - start t50 values indicated that the 8-oxodg lesion reduced the efficiency of hrev1 nucleotide incorporation by 6.8-fold ( table 2 ) . thus , of the four human y - family enzymes , hrev1 was most significantly inhibited by the 8-oxodg lesion relative to undamaged dg . however , due to the relatively fast rate of nucleotide incorporation of this enzyme , hrev1 was the second most efficient enzyme to bypass 8-oxodg overall and only 1.5-fold less efficient than hpol ( table 2 ) . to examine the ability of hrev1-generated lesion bypass products to be extended , the undamaged 21-mer/40-mer or damaged 21-mer/40-mer-8oxodg dna substrate was extended by the action of either hrev1 alone or in combination with hpol , the y - family enzyme purported to be specialized for the extension step of tls . within 30 s , hrev1 traversed the majority of the dg or 8-oxodg sites as demonstrated by the accumulation of the 22-mer product ( figure 2e and f ) . as previously observed in the running start primer extension assays , these 22-mer products were also not extended to full - length products by the action of hrev1 alone . however , if the control or damaged substrates were reacted with hrev1 for 30 s to allow for the bypass of the lesion or undamaged dg , followed by the addition of hpol to facilitate the extension of the hrev1-generated lesion bypass products , these 22-mer products were extended to full - length within 1 h. thus , although hrev1 is capable of bypassing the 8-oxodg lesion efficiently , a second polymerase is required for the extension of the lesion bypass products . standing start assays for hrev1 and hpol on undamaged and damaged dna templates . the position of the 8-oxodg lesion within the template strand is indicated by a y. a preincubated solution containing 400 nm of hrev1 and 100 nm of 5-[p]-radiolabeled ( b ) 21-mer/40-mer or ( c ) 21-mer/40-mer-8oxodg was rapidly mixed with a solution of all 4 dntps ( 200 m each ) . the reaction mixtures were quenched at the indicated times with 0.37 m edta and resolved by using denaturing page . the control dg or damaged 8-oxodg bypass% was plotted as a function of reaction time . a preincubated solution containing 100 nm of hrev1 and 100 nm of 5-[p]-radiolabeled ( e ) 21-mer/40-mer or ( f ) 21-mer/40-mer-8oxodg was rapidly mixed with a solution of all 4 dntps ( 200 m each ) . where indicated , hpol ( 300 nm ) was added to the reaction 30 s after the addition of the dntp solution . to examine the patterns and frequencies of mutations induced by tls of 8-oxodg catalyzed by the y - family dna polymerases , we used ht - sosa as summarized in scheme 1 . by using this high - throughput method , greater than two million nucleotide sequences generated from lesion bypass products were analyzed to provide a statistically robust data set ( table s2 , supporting information ) . as a control for errors induced by the high - throughput sequencing approach , we calculated the average error rates within a control flanking region ( positions 14 to 4 relative to the lesion site ) of each sequence , which was initially derived from the 13-mer or 16-mer primers ( table 1 and scheme 1 ) . the average base insertion , deletion , and substitution frequencies within this flanking region were found to be 0.014% , 0.055% , and 0.080% , respectively . these rates combined for an overall background error rate of 0.15 0.01% at each template position . this background error rate was at least 51-fold below the error rate of each enzyme opposite the lesion site and more than 10-fold below the background error rate at virtually every other position analyzed within the undamaged or damaged templates . as an additional control , using ht - sosa we calculated the average base substitution error rates for hpol , hpol , and hpol replicating the undamaged , control template to be 1.8 10 , 1.5 10 , and 1.5 10 , respectively . these values are similar to the nucleotide misincorporation fidelities of hpol ( 2.0 10 to 2.1 10 ) , hpol ( 3.5 10 to 2.9 10 ) , and hpol ( 9.3 10 to 1.1 10 ) as previously determined by presteady - state kinetic assays with undamaged dna substrates , and as previously measured by ht - sosa . therefore , ht - sosa is a viable technique for the study of the mutagenic profiles induced by tls of 8-oxodg catalyzed by the error - prone y - family polymerases . we found that hpol correctly incorporated dctp opposite 8-oxodg in 54.5% of the lesion bypass sequences analyzed ( figure 3 ) . therefore , hpol was 15.7-fold more error prone opposite the lesion than opposite template dg . opposite 8-oxodg , hpol misincorporated datp ( 41.7% ) over misincorporating dttp ( 1.3% ) or dgtp ( 1.2% ) and rarely generated a base insertion ( 0.1% ) or deletion ( 1.3% ) mutation . interestingly , this result indicates that even though the nucleotide incorporation rate of hpol opposite undamaged dg and 8-oxodg was almost identical ( figure 1a and b , and table 2 ) , hpol was the second most error prone y - family enzyme opposite the lesion site . to compare the hpol error frequency opposite the 8-oxodg lesion to other template positions , we examined the errors generated by hpol both three template positions upstream and downstream from the lesion site ( figure 4a and b ) . this analysis demonstrated that the error rates of hpol were nearly unchanged ( within 1.6-fold ) at these template positions in the presence or absence of 8-oxodg ( tables s3 , s4 and s5 , supporting information ) . therefore , the lesion only influenced the error rate of hpol during incorporations opposite from 8-oxodg . comparison of the preferred actions of the human y - family polymerases opposite 8-oxodg or dg . the relative number of nucleotide incorporations , insertion mutations , or deletion mutations produced by each polymerase or polymerase combination opposite template ( a ) dg or ( b ) 8-oxodg are indicated . histogram of the relative errors generated by the human y - family dna polymerases as a function of template position . lesion bypass analysis for ( a and b ) hpol , ( c and d ) hpol , ( e and f ) hpol , and ( g and h ) a combination of all 4 y - family polymerases by using either the undamaged 17-mer/40-mer substrate or the damaged 17-mer/40-mer-8oxodg substrate . the relative number of base insertions ( striped bar ) , substitutions ( black bar ) , and deletions ( white bar ) as a percentage of the total dntp incorporations are indicated at each template position . y. the indicated template positions are relative to the 8-oxodg lesion site within the 40-mer-8oxodg template . of the four human y - family enzymes , hpol was the most error prone opposite 8-oxodg ( figure 3b ) , preferentially misincorporating datp ( 78.0% ) over correct dctp ( 18.5% ) . overall , we found that hpol was 14-fold more error prone opposite the lesion when compared to undamaged dg . interestingly , the hpol error rates at positions downstream from the lesion site were nearly unchanged between the damaged or control templates ( figure 4c and d ) , even though the nucleotide incorporation rate of hpol was reduced at these positions within the primer extension assays ( figure 1d ) . in contrast , at positions upstream from the lesion site , the frequency of base insertion mutations increased by an average of 3.6-fold in the presence of 8-oxodg ( tables s3 , s6 , and s7 , supporting information ) . however , these insertion mutation events were rare , occurring at an average frequency of only 0.16% , indicating that 8-oxodg seldom altered the fidelity of hpol before the lesion entered the polymerase active site . ht - sosa analysis indicated that hpol correctly incorporated dctp and incorrectly incorporated da opposite 8-oxodg in 65.8% and 21.7% of the sequences analyzed , respectively ( figure 3 ) . thus , even though 8-oxodg reduced the nucleotide incorporation efficiency of hpol to a greater extent than either hpol or hpol ( table 2 ) , hpol was less error - prone than hpol or hpol opposite the lesion . overall , hpol generated more errors than either hpol or hpol at nearly every template position ( figure 4e and f ) . the fidelity of hpol has been shown to be lower opposite template pyrimidines than template purines and to violate normal watson consistently , we observed the fidelity of hpol to be the lowest opposite template base dt followed by dc , dg , and finally da when replicating the undamaged template ( figure 4e ) and found hpol to preferentially incorporate dg opposite template dt ( figure s3 , supporting information ) . although hpol produced a significant number of frameshift mutations while replicating the undamaged control template , the average hpol base deletion rate at template positions downstream from the 8-oxodg site increased by an average of 3.0-fold ( tables s3 , s8 , and s9 , supporting information ) . this increase in the average base deletion error rate of hpol downstream from 8-oxodg indicates that the lesion perturbed nucleotide incorporation by hpol after the lesion exited the polymerase active site . to determine the frequencies and types of mutations induced by 8-oxodg bypass catalyzed by hrev1 , the control 16-mer/40-mer substrate or damaged 16-mer/40-mer-8oxodg substrate were first reacted with hrev1 to allow for hrev1-mediated incorporation opposite undamaged dg or the 8-oxodg lesion , respectively . then , because hrev1 is incapable of completely extending similar dna substrates ( figure 2e and f ) , hpol was subsequently added to extend the dna products to full - length for ht - sosa analysis ( figure 5 ) . of all the y - family polymerases investigated individually or in combination , we found the combination of hrev1 and hpol was the least error prone opposite 8-oxodg by inserting correct dctp ( 92.3% ) over incorrect datp ( 6.9% ) . importantly , the standing start primer extension assays indicated that approximately 15% of hrev1 formed unproductive complexes with the damaged 21-mer/40-mer-8oxodg dna substrate ( figure 2d ) . therefore , we hypothesize that hpol catalyzed a portion of these observed datp incorporation events after the liberation of the 21-mer/40-mer-8oxodg substrate from unproductively bound hrev1 . this finding suggests that the error rate of hrev1 opposite 8-oxodg may be even lower than the error frequency observed here . nevertheless , this result is consistent with the demonstrated activity of hrev1 as a specialized dctp transferase , preferentially inserting dctp opposite damaged or undamaged templating bases . thus , we conclude that hrev1 is the best suited y - family polymerase to catalyze error - free tls of 8-oxodg . interestingly , the combination of hrev1 and hpol produced a significant number of deletion mutations at template positions + 1 and + 2 ( tables s10 and s11 , supporting information ) . the majority of these deletion mutations are double base deletion mutations , suggesting that these double base deletion mutations arose after hrev1 incorporated two dc nucleotides into the primer strand , followed by realignment of the primer strand to loop out the + 1 and + 2 template positions , and subsequent extension by hpol. this proposed loop out mechanism is consistent with the previously reported ability of hpol to extend primers that contain limited sequence complementarity at the 3 primer terminus . histogram of the relative errors generated by the combination of hrev1 and hpol as a function of template position . sequence analysis of the lesion bypass products generated by the combination of hrev1 and hpol with ( a ) the undamaged 21-mer/40-mer substrate or ( b ) the 21-mer/40-mer-8oxodg substrate . the relative number of base insertions ( striped bar ) , substitutions ( black bar ) , and deletions ( white bar ) as a percentage of the total dntp incorporations are shown at each template position . the template bases are denoted , and the 8-oxodg lesion is represented as y. the indicated template positions are relative to the 8-oxodg lesion site within the 40-mer-8oxodg template . all four human y - family enzymes are present within the nucleus during s - phase . however , the question of how an individual y - family polymerase is recruited to a stalled replication complex in order to bypass a particular lesion in vivo remains unanswered . we hypothesize that the inherent properties of each polymerase , such as affinity for the lesion site and lesion bypass efficiency , are predictive of the potential for each enzyme to bypass a particular lesion in vivo . in order to establish a direct competition between the human y - family enzymes for 8-oxodg bypass , ht - sosa was performed with an equal molar concentration of all four dna polymerases ( figure 4 g and h ) . this combination of all four enzymes misincorporated datp ( 50.0% ) over correctly incorporating dctp ( 43.5% ) , misincorporating dgtp ( 1.2% ) or dttp ( 1.3% ) , or generating a deletion mutation ( 4.0% ) or an insertion mutation ( 0.6% ) . therefore , the fidelity of lesion bypass by the combination of all four human enzymes most closely resembled the 8-oxodg bypass fidelity of hpol alone ( tables s4 , s5 , s12 and s13 , supporting information ) . consistent with the running start primer extension assays ( figure 1i and j ) , we concluded that in the presence of all four human y - family polymerases , hpol catalyzed the majority of nucleotide incorporation events , including opposite the lesion site . therefore , even though hpol is not the best suited human y - family member to catalyze error - free tls of 8-oxodg , the properties hpol allow this polymerase to outcompete hpol , hpol , and hrev1 for bypass of 8-oxodg . the highly mutagenic 8-oxodg lesion is one of the most prevalent lesions induced by oxidative dna damage . in this article , we systematically compared the 8-oxodg bypass efficiencies and fidelities of all four human y - family dna polymerases for the first time in order to predict the enzyme(s ) that have evolved to catalyze tls of 8-oxodg in vivo . we conclude that ( 1 ) hpol outcompetes hpol , hpol , and hrev1 for nucleotide incorporations opposite 8-oxodg in vitro ; ( 2 ) hpol is the only y - family member to traverse 8-oxodg with the same efficiency as undamaged dg ; ( 3 ) the efficiency of nucleotide incorporation by hrev1 is most significantly reduced opposite the lesion when compared to incorporations opposite undamaged dg ; ( 4 ) hrev1 fails to extend 8-oxodg lesion bypass products ; and ( 5 ) the combination of hrev1 to bypass 8-oxodg and a second polymerase to extend the lesion bypass product is the most accurate polymerase combination to perform tls of 8-oxodg , followed by hpol , hpol , and hpol individually . the relative 8-oxodg bypass efficiencies based upon the calculated t50 values of the human y - family polymerases are hpol > hrev1 hpol hpol ( table 2 ) . these findings agree with a previous report investigating 8-oxodg bypass by hpol , hpol , and hpol by using a gapped dna substrate strand displacement assay , although hrev1 was not included in that study . importantly , we found that the ability of hrev1 to perform the extension step of tls was almost completely eliminated by 8-oxodg ( figure 2c and f ) . therefore , even though 8-oxodg is efficiently bypassed by hrev1 , a switch to a second polymerase is required for the extension step . in fact , this inherent inability of hrev1 to extend the lesion bypass products may promote polymerase switching in vivo . if the switching does not occur , the extension of any lesion bypass product by hrev1 would result in one or more dctp incorporations into dna due to the function of hrev1 as a dctp transferase , likely leading to the substitution or frameshift mutations . thus , we hypothesize that the mostly error - free extension of lesion bypass products generated by hrev1 would be carried out in vivo by a second polymerase , such as hpol or dna polymerase , a b - family polymerase that is also suitable for the extension step of tls . by using ht - sosa , we found that the most common mutation induced by 8-oxodg bypass catalyzed by all four human y - family polymerases either alone or in combination was the incorporation of da opposite 8-oxodg ( figure 3b ) . these results are consistent with previous kinetic studies indicating the predominant substitution catalyzed by hpol , hpol , and hpol opposite 8-oxodg is the misincorporation of datp . this miscoding potential of 8-oxodg is strongly influenced by the ratio of the anti and syn conformations of 8-oxodg within a polymerase active site . interestingly , this dual coding potential of lesions such as 8-oxodg is irrelevant to nucleotide incorporations by hrev1 , as structural studies indicate this enzyme uses a novel mechanism whereby incoming dctp interacts and forms two hydrogen bonds with the side chain of arg357 within the n - digit domain of hrev1 , rather than the templating base . this dctp transferase activity is also supported by kinetic analysis demonstrating hrev1 preferentially incorporates dctp opposite any templating base , damaged or undamaged . here , we found that the combination of hrev1 and hpol was nearly error - free opposite 8-oxodg ( figure 3b ) . indeed the low frequency of datp incorporation ( 6.9% ) detected by ht - sosa may be due to the displacement of unproductively bound hrev1 by active hpol , which in turn bypassed the lesion and predominantly incorporated datp opposite 8-oxodg . therefore , hrev1 may be almost completely error free when bypassing 8-oxodg . in this study , we demonstrate that when all four human y - family polymerases are tested in combination , hpol outcompetes hpol , hpol , and hrev1 for tls of 8-oxodg ( table 2 ) . notably , previous studies indicate that among the human y - family polymerases , hpol possesses the highest efficiency of nucleotide incorporation into undamaged dna substrates and a 2- to 5-fold greater affinity for undamaged dna substrates when compared to that of hpol , hrev1 , or hpol. furthermore , it has been shown that the presence of 8-oxodg as a templating base does not reduce the binding affinity of either yeast pol or the model y - family polymerase dpo4 for a dna substrate , suggesting that the y - family polymerases are well suited to accommodate this small lesion within their relatively large active sites , without a reduction in dna binding capacity . thus , the superior dna binding affinity of hpol likely contributes to its dominance within the four enzyme combination assays ( figures 1i and j ) . however , differences in dna binding affinity alone are not sufficient to explain the 100- to 100,000- fold difference in the t50 values between hpol and the other three y - family dna polymerases ( table 2 ) . instead , we conclude that a superior nucleotide incorporation efficiency of hpol is the primary contributor to the dominance of hpol for 8-oxodg bypass . importantly , we chose to test the abilities of the human y - family polymerases to bypass 8-oxodg at equal molar concentrations in order to gain insight into the intrinsic abilities of these polymerases to compete for tls of 8-oxodg . however , the relative in vivo expression profiles of the human y - family polymerases under conditions of oxidative dna damage are not known . indeed the expression of each of the y - family polymerases may not be similar between cell types or may be dynamic during the cell cycle . further studies are especially needed to elucidate the regulation of the expression and activities of the y - family polymerases in response to cellular dna damage in vivo , which remains an important , unanswered question . eukaryotes utilize a number of mechanisms to reduce the mutagenic consequences of 8-oxodg , including ( 1 ) excision of the damaged base from 8-oxodgdc pairs by 8-oxoguanine dna glycosylase 1 ( ogg1 ) to initiate base excision repair ( ber ) , ( 2 ) excision of the da base from 8-oxodg : da mispairs by myh adenine glycosylase to facilitate a new incorporation event opposite the lesion , and ( 3 ) hydrolysis of 8-oxodgtp to 8-oxodgmp by mth1 to prevent incorporation of the damaged dntp into replicating dna . despite these important repair mechanisms , 8-oxodg lesions do persist during s - phase and are encountered by the replication fork . the majority of mutations induced by 8-oxodg in vivo are g t transversion mutations , indicating that the error - prone tls of 8-oxodg principally results in datp misincorporation . our finding that hpol outcompetes hpol , hpol , and hrev1 for nucleotide incorporation opposite 8-oxodg is most consistent with a model wherein hpol is the predominant y - family member to catalyze the error - prone bypass of 8-oxodg in vivo . however , in the absence of functional pol , another y - family polymerase may catalyze the bypass 8-oxodg . consistently , plasmid - based mutation assays conducted within wild - type or hpol-deficient human cell lines demonstrated that in the absence of hpol , the action of an unknown polymerase increased the total 8-oxodg - induced mutation frequency by 1.2- to 2-fold , primarily due to an increase in g t transversion mutations . our data suggest that hpol is the unknown polymerase since it is the only y - family enzyme which misincorporates datp opposite 8-oxodg with a higher frequency than hpol ( figure 3b ) . the elucidation of which tls - specialized polymerases are responsible for the error - free or error - prone bypass of specific types of dna lesion sites in vivo remains an unanswered and challenging question . here , we used primer extension assays paired with the recently established ht - sosa approach to directly compare the efficiency and fidelity of 8-oxodg bypass catalyzed by each of the human y - family polymerases individually and in combination . overall , our data indicate that even though hpol is error - prone opposite 8-oxodg , the superior efficiency of hpol for tls of 8-oxodg suggests that this enzyme is the primary y - family enzyme to catalyze the bypass of 8-oxodg in vivo . however , we can not completely rule out a role for the other three y - family members for tls of 8-oxodg in vivo . indeed , many dna polymerases may catalyze the bypass of 8-oxodg lesions in vivo to different extents . the comprehensive biochemical studies reported here will provide a basis for further evaluation of these possibilities by in vivo studies .

one of the most common lesions induced by oxidative dna damage is 8-oxo-7,8-dihydro-2-deoxyguanosine ( 8-oxodg ) . replicative dna polymerases poorly traverse this highly mutagenic lesion , suggesting that the replication fork may switch to a polymerase specialized for translesion dna synthesis ( tls ) to catalyze 8-oxodg bypass in vivo . here , we systematically compared the 8-oxodg bypass efficiencies and fidelities of the tls - specialized , human y - family dna polymerases eta ( hpol ) , iota ( hpol ) , kappa ( hpol ) , and rev1 ( hrev1 ) either alone or in combination . primer extension assays revealed that the times required for hpol , hrev1 , hpol , and hpol to bypass 50% of the 8-oxodg lesions encountered ( t50bypass ) were 0.58 , 0.86 , 108 , and 670 s , respectively . although hrev1 bypassed 8-oxodg efficiently , hrev1 failed to catalyze the extension step of tls , and a second polymerase was required to extend the lesion bypass products . a high - throughput short oligonucleotide sequencing assay ( ht - sosa ) was used to quantify the types and frequencies of incorporation errors produced by the human y - family dna polymerases at and near the 8-oxodg site . although hpol bypassed 8-oxodg most efficiently , hpol correctly incorporated dctp opposite 8-oxodg within only 54.5% of the sequences analyzed . in contrast , hpol bypassed the lesion least efficiently but correctly incorporated dctp at a frequency of 65.8% opposite the lesion . the combination of hrev1 and hpol was most accurate opposite 8-oxodg ( 92.3% ) , whereas hpol alone was the least accurate ( 18.5% ) . the t50bypass value and correct dctp incorporation frequency in the presence of an equal molar concentration of all four y - family enzymes were 0.60 s and 43.5% , respectively . these values are most similar to those of hpol alone , suggesting that hpol outcompetes the other three y - family polymerases to catalyze 8-oxodg bypass in vitro and possibly in vivo .

Introduction Experimental Procedures Results Discussion

PMC3897096

reconstruction of breast bu deep inferior epigastric perforator ( diep ) flap is popular throughout the world . however , we are still concerned about how to improve the aesthetic results . in this respect what we are still trying to improve is breast -symmetry in terms of volume and shape , bulkiness of the inferior lateral quadrant of the new breast ( if anastomosis were performed in axilla ) , loss of volume of the upper pole and lack of projection of the inferior pole . the aim of our work is to describe our personal techniques in order to improve the final aesthetic outcome in diep flap breast reconstruction . since 1997 , we have been performing 226 breast reconstructions with diep flap in the department of plastic and reconstructive surgery at s. orsola - malpighi hospital . the mean average age of our patients was 49.6 years old ( range : 29 - 73 ) . the reconstruction was immediate in 96 cases ( 42 , 48% ) and delayed in 130 cases ( 57 , 52% ) ; in this last group 16 patients were previously reconstructed using other techniques ( 11 by prosthesis and 5 with lipofilling ) . most of these refinements were performed intraoperatively , but when this is not possible , we perform secondary revisions a few months later , usually under local anaesthesia . during the flap insetting , we usually leave the lateral part of the flap bulky in order not to have any risk with flap vascularisation . during the flap modeling , we harvest a subcutaneous pocket superiorly , in order to fill the upper pole of the new breast with the flap , partially de - epithelialised and fixed to the pectoral major muscle with re - absorbable stitches . in order to reduce the risk of secondary liponecrosis , we usually try not to remove too much fat from the adipose layer of the superior edge of the flap . this can sometimes lead to a gap between the diep flap and the skin in the upper pole , particularly evident in obese patients where the abdominal fat layer is very thick . when it is possible , we try to correct lateral bulkiness and the upper pole gap at the same time harvesting local flaps or using a combination of lipoaspiration and lipofilling . local flaps are our first choice . under local anaesthesia , we harvest a vertical flap , based superiorly , from the new breast 's lateral side , exactly where the bulkiness is . the flap is then de - epithelialised and transposed to a subcutaneous pocket in the upper pole where the defect is . the flap is fixed with transcutaneous stitches and the donor site is closed primarily [ figure 1 ] . the flap was de - epithelialised and transposed to a subcutaneous pocket in the upper pole our second choice is to make a lipoaspiration of the lateral bulky side and use this tissue , harvested following the principles described by coleman , to lipofill the upper pole . in many cases , the amount of adipose tissue obtained from the bulky area is not sufficient , so we use other donor sites . in young patients with a projected contralateral breast or after mastopexy , we often need to improve diep flap projection in order to obtain a better symmetry . the lack of projection can easily be solved by recreating the inframammary fold in the reconstructed breast as we usually do with implants breast reconstruction . the position of the fold is decided after abdominal donor site closure to avoid tension effects induced by abdominoplasty . in delayed reconstructions , flap projection improvement can be reached using local flaps . during diep flap modelling , the inferior margin of the flap slides at the level of the inframammary fold we started to use this skin paddle to harvest a small flap that is de - epithelialised and pedicled inferiorly at the level of the inframammary crease . the flap pedicle is exactly positioned at a sternal distance corresponding to the other breast major projection point . then the flap is rolled on itself to reproduce a cone like shape [ figure 2 ] . the pedicle was centred on the major projection point and the flap was sutured on itself to obtain a cone shape this is a challenging technique to obtain a good projection , especially in young patients with a projected contralateral breast that does not need mastopexy . we performed this reconstruction in a 36-year - old female patient who affected by a recurrence of a mucinous carcinoma of the right breast . our surgical plan was to harvest diep flap and contralateral breast reduction , but the patient was not prepared to accept any contralateral breast scar . as the patient was slightly overweight , the abdomen was sufficient to harvest a double diep flap to reconstruct one breast , in order to give it more volume and projection . during the flap harvesting , we found two good perforators , one on the right side of the abdomen and the other one on the left side , near the lateral border of the rectus abdominis muscle . we harvested two distinguished flaps : areas 1 - 3 composed the left side flap and the right side flap was composed of area 4 . the smaller flap was v - shaped and de - epithelialised obtaining a cone , similar in shape to a prosthesis ; flap modeling was made before pedicle section . the two flaps were then transferred and immediately vascularised with end - to - end anastomosis to thoracodorsal vessels , for the bigger one and circumflex scapular vessels , for the buried flap . the smaller flap was buried under the other to obtain an increased volume and a better projection . the inframammary fold was fixed with a 2/0 polypropylene non - reabsorbable suture and the lower side of the bigger flap was sutured to the thorax at this level [ figure 3 ] . monolateral double deep inferior epigastric perforator flap : the small one was v - shaped and sutured on itself on the medial edge obtaining a shape similar to prosthesis . the final effect was the increase of volume and projection of the reconstructed breast breast asymmetry is always observed in unilateral breast reconstruction . more frequently the contralateral breast is bigger and needs to be reduced . rarely , especially in young patients , the contralateral breast can be hypoplastic and a second diep flap can be used like an auto - prosthesis for breast augmentation , thus avoiding the need of an implant . the flap used for breast reconstruction included areas 1 - 3 of diep flap and is transferred and modelled as usual . a smaller flap corresponding to area 4 , based on a different perforator , is de - ephitelialised and sutured on itself medially to obtain a cone similar in shape to prosthesis . , a sub - glandular pocket is harvested on the side of breast augmentation and the flap is inserted in it and fixed to the major pectoralis muscle with reabsorbable stitches . the anastomoses are performed in the axilla , leading the pedicle through a subcutaneous tunnel , to the thoracodorsal or circumflex scapular vessels [ figure 4 ] . double deep inferior epigastric perforator flap for contralateral breast augmentation : the two flaps were harvested based on the two deep inferior epigastric pedicles . the small flap was inserted in a subglandular pocket through an inframammary access during the flap insetting , we usually leave the lateral part of the flap bulky in order not to have any risk with flap vascularisation . during the flap modeling , we harvest a subcutaneous pocket superiorly , in order to fill the upper pole of the new breast with the flap , partially de - epithelialised and fixed to the pectoral major muscle with re - absorbable stitches . in order to reduce the risk of secondary liponecrosis , we usually try not to remove too much fat from the adipose layer of the superior edge of the flap . this can sometimes lead to a gap between the diep flap and the skin in the upper pole , particularly evident in obese patients where the abdominal fat layer is very thick . when it is possible , we try to correct lateral bulkiness and the upper pole gap at the same time harvesting local flaps or using a combination of lipoaspiration and lipofilling . local flaps are our first choice . under local anaesthesia , we harvest a vertical flap , based superiorly , from the new breast 's lateral side , exactly where the bulkiness is . the flap is then de - epithelialised and transposed to a subcutaneous pocket in the upper pole where the defect is . the flap is fixed with transcutaneous stitches and the donor site is closed primarily [ figure 1 ] . the flap was de - epithelialised and transposed to a subcutaneous pocket in the upper pole our second choice is to make a lipoaspiration of the lateral bulky side and use this tissue , harvested following the principles described by coleman , to lipofill the upper pole . in many cases , the amount of adipose tissue obtained from the bulky area is not sufficient , so we use other donor sites . in young patients with a projected contralateral breast or after mastopexy , we often need to improve diep flap projection in order to obtain a better symmetry . the lack of projection can easily be solved by recreating the inframammary fold in the reconstructed breast as we usually do with implants breast reconstruction . the position of the fold is decided after abdominal donor site closure to avoid tension effects induced by abdominoplasty . in delayed reconstructions , flap projection improvement can be reached using local flaps . during diep flap modelling , the inferior margin of the flap slides at the level of the inframammary fold we started to use this skin paddle to harvest a small flap that is de - epithelialised and pedicled inferiorly at the level of the inframammary crease . the flap pedicle is exactly positioned at a sternal distance corresponding to the other breast major projection point . then the flap is rolled on itself to reproduce a cone like shape [ figure 2 ] . the pedicle was centred on the major projection point and the flap was sutured on itself to obtain a cone shape this is a challenging technique to obtain a good projection , especially in young patients with a projected contralateral breast that does not need mastopexy . we performed this reconstruction in a 36-year - old female patient who affected by a recurrence of a mucinous carcinoma of the right breast . our surgical plan was to harvest diep flap and contralateral breast reduction , but the patient was not prepared to accept any contralateral breast scar . as the patient was slightly overweight , the abdomen was sufficient to harvest a double diep flap to reconstruct one breast , in order to give it more volume and projection . during the flap harvesting , we found two good perforators , one on the right side of the abdomen and the other one on the left side , near the lateral border of the rectus abdominis muscle . we harvested two distinguished flaps : areas 1 - 3 composed the left side flap and the right side flap was composed of area 4 . the smaller flap was v - shaped and de - epithelialised obtaining a cone , similar in shape to a prosthesis ; flap modeling was made before pedicle section . the two flaps were then transferred and immediately vascularised with end - to - end anastomosis to thoracodorsal vessels , for the bigger one and circumflex scapular vessels , for the buried flap . the smaller flap was buried under the other to obtain an increased volume and a better projection . the inframammary fold was fixed with a 2/0 polypropylene non - reabsorbable suture and the lower side of the bigger flap was sutured to the thorax at this level [ figure 3 ] . monolateral double deep inferior epigastric perforator flap : the small one was v - shaped and sutured on itself on the medial edge obtaining a shape similar to prosthesis . the lack of projection can easily be solved by recreating the inframammary fold in the reconstructed breast as we usually do with implants breast reconstruction . the position of the fold is decided after abdominal donor site closure to avoid tension effects induced by abdominoplasty . in delayed reconstructions , flap projection improvement can be reached using local flaps . during diep flap modelling , the inferior margin of the flap slides at the level of the inframammary fold we started to use this skin paddle to harvest a small flap that is de - epithelialised and pedicled inferiorly at the level of the inframammary crease . the flap pedicle is exactly positioned at a sternal distance corresponding to the other breast major projection point . then the flap is rolled on itself to reproduce a cone like shape [ figure 2 ] . the pedicle was centred on the major projection point and the flap was sutured on itself to obtain a cone shape this is a challenging technique to obtain a good projection , especially in young patients with a projected contralateral breast that does not need mastopexy . we performed this reconstruction in a 36-year - old female patient who affected by a recurrence of a mucinous carcinoma of the right breast . our surgical plan was to harvest diep flap and contralateral breast reduction , but the patient was not prepared to accept any contralateral breast scar . as the patient was slightly overweight , the abdomen was sufficient to harvest a double diep flap to reconstruct one breast , in order to give it more volume and projection . during the flap harvesting , we found two good perforators , one on the right side of the abdomen and the other one on the left side , near the lateral border of the rectus abdominis muscle . we harvested two distinguished flaps : areas 1 - 3 composed the left side flap and the right side flap was composed of area 4 . the smaller flap was v - shaped and de - epithelialised obtaining a cone , similar in shape to a prosthesis ; flap modeling was made before pedicle section . the two flaps were then transferred and immediately vascularised with end - to - end anastomosis to thoracodorsal vessels , for the bigger one and circumflex scapular vessels , for the buried flap . the smaller flap was buried under the other to obtain an increased volume and a better projection . the inframammary fold was fixed with a 2/0 polypropylene non - reabsorbable suture and the lower side of the bigger flap was sutured to the thorax at this level [ figure 3 ] . monolateral double deep inferior epigastric perforator flap : the small one was v - shaped and sutured on itself on the medial edge obtaining a shape similar to prosthesis . rarely , especially in young patients , the contralateral breast can be hypoplastic and a second diep flap can be used like an auto - prosthesis for breast augmentation , thus avoiding the need of an implant . the flap used for breast reconstruction included areas 1 - 3 of diep flap and is transferred and modelled as usual . a smaller flap corresponding to area 4 , based on a different perforator , is de - ephitelialised and sutured on itself medially to obtain a cone similar in shape to prosthesis . , a sub - glandular pocket is harvested on the side of breast augmentation and the flap is inserted in it and fixed to the major pectoralis muscle with reabsorbable stitches . the anastomoses are performed in the axilla , leading the pedicle through a subcutaneous tunnel , to the thoracodorsal or circumflex scapular vessels [ figure 4 ] . double deep inferior epigastric perforator flap for contralateral breast augmentation : the two flaps were harvested based on the two deep inferior epigastric pedicles . the small flap was inserted in a subglandular pocket through an inframammary access the average time of surgery is about 7 h. only in double diep flap the time is slightly longer ( average time : 9 h ) . hospitalisation was the same in all cases ( average 8 days , range : 7 - 9 ) . with regard to major complications , in this series , we observed 9 cases of total necrosis of the flap and 15 cases of partial necrosis . no complications , such as necrosis or loss of volume , were observed in patients treated by local lateral flaps . this means lack of softness of the new breast . in the early treated cases , we observed liponcreosis in 20% of flaps while in the last ones we observed a failure rate decreasing ( < 5% ) due to a better planning and particularly to a reduction of the height / length ratio of the lateral flaps . the results were obtained in patients treated with the presented techniques were good in terms of symmetry and shape of the breasts with a high level of satisfaction reported by patients . this last consideration may not be indicative because also patients treated using a traditional method usually reported a similar level of satisfaction . improvement of lateral bulkiness and upper pole deficiency could be easily achieved using local flaps or lipo - scuplture . these two methods allow correction of both defects at the same time , with a simple surgery under local anaesthesia . we usually plan these corrections at an average time of 6 months after primary reconstruction , when the result can be considered as quite stable . we performed these techniques in almost 40% of our patients and very often we combined the two techniques together . we usually harvest the local lateral flap even if it 's almost always not sufficient to bridge the gap because it 's a simple procedure under local anaesthesia , which use tissues that other ways will be thrown away . on the other hand , in our experience , the lateral bulkiness can be better corrected with tissue excision than liposuction ; this is due to a certain degree of fibrosis developed in this region after surgery . improvement of lower pole projection has two main goals : better shape of the new breast and better symmetry with contralateral breast . applying both inframammary suture and local flaps we noticed no significant increase in surgical time . with a better planning of the base / high ratio of the flap we were been able to reduce the incidence of partial liponecrosis from 20% to 5% of cases . incidentally , no major complications like infection or fistulae due to liponecrosis were observed , but only a lack of softness of the inferior pole . the results obtained with the use of local flaps alone are not satisfactory , but this is a very simple technique , with no adjunctive costs for the patients , which can be associated to the inframammary fold recreation improving the results . we have used this technique in all cases of delayed reconstruction since 2010 . in our experience , unilateral reconstruction and contralateral breast augmentation with double diep flap could be useful and innovative . area 4 could be harvested as a free flap on its pedicle , modelled and used as autoprosthesis for contralateral augmentation . we used this technique in five cases : two immediate and three delayed reconstructions after failure of reconstruction with prosthesis . addictive mammaplasty with diep flap is indicated in all cases of young patients with hypoplastic contralateral breast or in patients that required breast augmentation with no implants . the inframammary access used for flap in setting is not evident as in a common augmentation mammoplasty and the same is for the scar made in the axilla for anastomosis . area 4 based on its pedicle , we obtained a flap that could be easily modelled as an auto - prosthesis and buried under the other flap . we think that this method could be indicated in patients with a large contralateral breast when it is not possible to re - shape it for oncological reasons or to respect the wishes of the patient . based on the above study it can be concluded that microsurgical breast reconstruction has almost reached optimum standards both in terms of surgical techniques and aesthetic results . the aim of autologous tissue breast reconstruction is to achieve a good aesthetic result without implants . in our work , we describe our personal techniques in order to improve the final aesthetic outcome in diep flap breast reconstruction . our experience with refinements like the use of local flaps in delayed reconstructions or the inframammary fold recreation to increase projection is an example of how you can save surgery time and costs , achieving increasingly better aesthetical results in the very first breast reconstruction . lateral bulkiness and upper pole deficiency can easily be corrected at the same time , under local anaesthesia and with a very little additional sacrifice for the patient . unilateral or bilateral breast reconstruction with double diep flap could be a valuable option to consider when indicated . no major complications were reported and post - operative recovery was not affected by the procedure . any increase in surgery time is related to additional complete pedicle dissection and anastomosis . in our opinion , this disadvantage is not sufficient to avoid using this procedure that must be considered as a valuable option in selected cases .

background : now - a - days , deep inferior epigastric perforator ( diep ) flap breast reconstruction is widespread throughout the world . the aesthetical result is very important in breast reconstruction and its improvement is mandatory for plastic surgeons.materials and methods : the most frequent problems , we have observed in breast reconstruction with diep flap are breast asymmetry in terms of volume and shape , the bulkiness of the inferior lateral quadrant of the new breast , the loss of volume of the upper pole and the lack of projection of the inferior pole . we proposed our personal techniques to improve the aesthetical result in diep flap breast reconstruction . our experience consists of more than 220 diep flap breast reconstructions . results : the methods mentioned for improving the aesthetics of the reconstructed breast reported good results in all cases.conclusion:the aim of our work is to describe our personal techniques in order to correct the mentioned problems and improve the final aesthetical outcome in diep flap breast reconstruction .

INTRODUCTION MATERIALS AND METHODS SURGICAL TECHNIQUES Lateral bulkiness reduction and upper pole improvement Flap projection improvement Inframammary fold recreation Local flaps in delayed reconstructions Double DIEP flap in monolateral reconstruction Breast symmetry RESULTS DISCUSSION CONCLUSION

PMC3270402

the prevalence and severity of obesity is increasing dramatically among children and adolescents in many parts of the world , whereas prevalence rates are estimated to increase in the next decades . in children , excess body fat appears to be strongly associated with the clustering of risk factors , such as hyperglycemia , dyslipidemia , and hypertension , which play a key role in the pathogenesis of the metabolic syndrome ( mets ) . obesity and the mets risk in children have been recently associated with systemic inflammatory markers , in particular c - reactive protein ( crp ) [ 3 , 4 ] , implying that low - grade inflammation can already exist in childhood and may be a potential link between the obesity and the mets . among behavioral variables , cardiorespiratory fitness has a protective role in mets and inflammatory factors ; however , it is not entirely clear if the interrelations among cardiorespiratory fitness , mets risk , and inflammation in children are independent or partly due to the mediating effect of obesity , since the existing data are limited and equivocal [ 5 , 6 ] . recent evidence indicates that the prevalence rates of childhood obesity in greece remain high [ 1 , 7 ] and often coexist with low cardiorespiratory fitness and an unfavorable cardiometabolic risk profile . for the greek pediatric population these data suggest an increased cardiovascular morbidity in adulthood , given that high - risk children and adolescents are likely to become high - risk adults . although the relationship among obesity and dyslipidemia in greek children has been thoroughly investigated [ 9 , 11 ] , there is a paucity of data regarding the clustering of metabolic risk factors , inflammation , and their relationship with cardiorespiratory fitness . the present study was undertaken in an attempt to investigate the prevalence of the mets and examine the associations among cardiorespiratory fitness , mets risk , and crp in 11-year - old children . this study sample consisted of 112 pupils ( 54 females , 58 males , mean age 11.4 0.4 years ) , registered in the sixth grade of 15 randomly selected primary schools in north attica , greece . subjects were healthy at the time of investigation and had no chronic or acute illnesses . prior to the commencement of the study , permission from the school principal was obtained and parental consent was secured after full explanation of study objectives and data collection procedures . the research ethics committee of democritus university of thrace approved the investigation and permission was granted by the greek ministry of education . measures of anthropometry and fitness were conducted in the school environment ( during pe classes ) by the same experienced investigator ( the principal author ) , using the same order of testing procedures and under similar conditions . blood samples and blood pressure data were obtained at the same clinical biochemistry accredited laboratory by a medical practitioner . students were weighed on an electronic scale to the nearest 0.5 kg ( seca beam balance 710 ) , lightly dressed and barefooted . standing height was measured to the nearest 0.5 cm ( seca stadiometer 208 ) with each subject 's shoes off , feet together , and head in the frankfort horizontal plane . body mass index ( bmi ) bmi values were also converted to standard deviation scores ( sdss ) , whereas the iotf 's age- and gender - specific bmi cut - off points were used to calculate the prevalence rates for overweight / obesity . waist circumference , measured at the level of the umbilicus with a tape measure to the nearest 0.1 cm , was used as an indicator of central obesity . cardiorespiratory fitness was assessed by using the 20 m multistage shuttle run test , a standard field test included in the european fitness test battery . in brief , 510 subjects performed a series of runs across a 20 m track , changing direction at the end of each run to coincide with an audio signal that was getting progressively faster . subjects started running at a speed of 8.5 km / hr , and speed increased at various stages ( 0.5 km / hr every minute ) . each stage was made up of several shuttle runs , and pupils were instructed to keep pace with the signals as long as possible . the number of laps fully completed were recorded for each pupil and subsequently used to estimate cardiorespiratory fitness as predicted maximal oxygen uptake ( vo2max ) in ml / kg / min . based on gender- and age - specific cut - off values of vo2max participants were classified into two fitness groups . blood pressure ( bp ) was measured in the right arm , using a mercury sphygmomanometer with appropriate size cuffs and cuff bladders , after the subject had been sitting quietly for at least 5 min . the mean of two measurements at the first and the fifth korotkoff phase was recorded for systolic and diastolic bp , respectively . blood samples were obtained from each subject early in the morning , following an overnight fast . blood was centrifuged for plasma separation , and 1.5 ml aliquots were pipetted into plastic eppendorf tubes and stored at 70c until further analyses . plasma glucose , triglycerides , and hdl cholesterol ( hdl - c ) were determined using chromatographic enzymatic method on an automated mb ii analyzer ( dade behring , marburg , germany ) . crp was measured by high - sensitive latex - enhanced immunoassay on a bn ii nephelometer from the same company . the mets was defined as meeting 3 of the following age- and gender - specific criteria : waist circumference 90th percentile , glucose 100 mg / dl , triglycerides 100 mg / dl , hdl - c < 50 mg / dl , and systolic or diastolic bp 90th percentile [ 17 , 18 ] . we further computed a mets risk score as follows : for each of the mets components , an absolute z score was computed . for hdl - c the z score was multiplied by 1 to indicate higher metabolic risk with increasing value . the z scores of systolic and diastolic bp were averaged and then added to the rest of the z scores . this risk score is statistically more sensitive and less error - prone by comparison to dichotomous approaches . we also computed a nonobesity mets risk score , omitting the z score from waist circumference . because of a nonnormal distribution , crp values were logarithmically transformed prior to the analyses . continuous variables are presented as mean values standard error of means , while qualitative variables are presented as absolute and relative frequencies . relationships among cardiorespiratory fitness , mets risk score , and crp levels were summarized with pearson 's correlations . the independent effect of low fitness level on anthropometric indices , metabolic characteristics , mets risk score , and crp was assessed using one - way ancova . descriptive characteristics of the subjects by gender and for the entire sample are presented in table 1 . anthropometric parameters are in line with recently published paediatric data [ 1 , 7 , 8 ] , suggesting that the sample is representative of periadolescent children in greece . mean bmi values , mean plasma concentrations of the mets components , and mean bp recordings were within acceptable ranges and did not differ between genders . sds - bmi was significantly higher in males compared to females ( t(110 ) = 2.52 , p = 0.013 ) . among all participants , 34.8% had one component , 12.5% had two components , 2.7% had three components , 1.8% had four components , and 0.9% had five components of the mets . hyperglycemia was the most common metabolic abnormality in both genders ( 27.7% ) , followed by hypertriglyceridemia ( 17.9% ) . elevated bp was more frequent in females , but differences did not achieve statistical significance ( = 3.43 , p = 0.064 ) . the prevalence of the mets was 5.4% , without statistically significant gender differences ( = 0.008 , p = 0.928 ) . pearson 's correlation analyses revealed that cardiorespiratory fitness was inversely associated with the mets risk score ( r = 0.386 , p < 0.001 ) , and crp ( r = 0.394 , p < 0.001 ) . mets risk score and crp were positively interrelated ( r = 0.544 , p < 0.0005 ) . a high percentage of the study participants demonstrated cardiorespiratory fitness values considerably lower than the recommended fitness levels for metabolic health . in particular , ~72% of both genders did not achieve the acceptable fitness values for children of comparable age . children with lower fitness levels , compared to their peers with increased fitness levels , demonstrated significantly increased bmi ( 21.01 versus 17.91 kg / m , f(1,111 ) = 17.55 , p < 0.001 ) , sds - bmi ( 1.07 versus 0.14 , f(1,111 ) = 13.43 , p < 0.001 ) and waist circumference ( 72.89 versus 64.23 cm , f(1,111 ) = 19.82 , p < 0.001 ) . in addition , pupils in the lower fitness group showed significantly higher mets risk score ( f(1,93 ) = 13.03 , p < 0.001 ) and crp values ( f(1,93 ) = 7.67 , p < 0.007 ) , compared to the high - fitness group ( figure 1 ) . adjustment for central obesity attenuated the observed differences in the mets risk score , which were no longer significant ( figure 1(a ) ) . differences in crp values between fitness groups decreased also when adjusted for waist circumference ( figure 1(b ) ) ; however , they remained borderline significant ( f(1,93 ) = 3.49 , p < 0.067 ) . analyses including bmi or sds - bmi instead of waist circumference yielded similar results ( data not shown ) . the mets has a multifactorial aetiology , and the mechanisms underlying its onset are not completely understood . in the present study we investigated the prevalence of the mets and examined the independent associations among cardiorespiratory fitness , mets risk , and crp in greek children . using a paediatric definition based closely on atp iii [ 17 , 18 ] , we found that the prevalence of mets in our sample was 5.4% . this prevalence rate is similar to those reported in us and french adolescents ( 45.8% ) [ 2123 ] but lower than the prevalence reported in iranian paediatric populations ( 14.1% ) . these data indicate that the mets is a common health problem among different paediatric populations , underlining the need for effective policies and appropriate intervention strategies at global , national , and regional level . we further found that the protective effect of cardiorespiratory fitness on the mets risk score was explained by the confounding effect from central obesity . our results are in agreement with previous findings , showing that cardiorespiratory fitness is not independently associated with markers of the mets . however , recent data from the european youth heart study ( eyhs ) [ 19 , 25 , 26 ] and the national health and nutrition examination survey ( nhanes ) 19992002 revealed that cardiorespiratory fitness is a significant and independent predictor of the mets in children , whereas the benefits of higher cardiorespiratory fitness seem to extend also to children at lower metabolic risk . differences in body composition and fitness levels are known to affect the metabolic profile in children and could explain at least part of these discrepancies among studies . compared to their north european and north american age - related peers , greek children exhibit higher rates of overweight / obesity and worse cardiorespiratory fitness performance [ 8 , 19 , 27 , 2931 ] . these findings suggest that children in the present study did not achieve the required threshold in cardiorespiratory fitness , in order to profit from its beneficial effects with respect to the risk of mets in the face of obesity . indeed , nassis et al . have shown that exercise - related cardiorespiratory fitness enhancement in overweight girls can lead to marked improvements in insulin sensitivity , without changes in body composition . in addition , even if central obesity is an intermediate link between low fitness and mets risk , fitness will still be the principal cause , even if its association with mets risk disappears after adjustment for an intermediate variable in the causal chain ( i.e. , central obesity ) . in line with a previous report in children and young adults , we found that the association between cardiorespiratory fitness and crp was largely explained by their interrelation to obesity . reported no relationship between crp and cardiorespiratory fitness in 12 - 13-years - old schoolchildren . in contrast , results from the ten towns children 's study and more recent data from south african children reported strong correlations between fitness and crp . an explanation for this discrepancy may be the fact that in the ten towns children 's study a crude estimate of physical fitness ( i.e. , resting heart rate ) was used , whilst in the latter study the investigators did not take into account the confounding effect of obesity . more studies are needed to improve our understanding of the interrelations among cardiorespiratory fitness , crp , and obesity in children . our findings indicate that the mechanisms linking cardiorespiratory fitness , mets risk , and crp are extensively affected by obesity . further , the favourable effect of cardiorespiratory fitness on mets risk may not become evident when fitness levels underlie a critical threshold . given that high levels of cardiorespiratory fitness improve the metabolic state of obese children , attenuate the proinflammatory milieu associated with obesity , and offer protection against the development of the insulin resistance syndrome from childhood into adolescence and young adulthood [ 3739 ] , children should be encouraged to achieve the recently recommended physical activity levels ( 90 min / day of at least moderate intensity ) , in order to prevent clustering of cardiovascular disease risk factors and improve their metabolic and overall health profile . a root cause in the development of the mets is insulin resistance , which was not assessed in the present study . however , epidemiologic studies indicate that a substantial proportion of subjects with the mets do not have evidence of insulin resistance and the correlation between insulin resistance and individual components of the mets is weak to moderate . however , it is unlikely that our findings are invalidated by the use of the 20 m multistage shuttle run test since it has been extensively used in the literature and is recommended for paediatric studies as reliable , valid , and cost - effective [ 8 , 13 , 14 , 30 , 31 ] . further , a strict random sampling of all eligible pupils was virtually impossible , given that the study was dependent on the principals ' disposal and the willingness of individual children and their parents to participate . the prevalence of overweight / obesity , however , was similar to that reported for school - aged children from other greek regions [ 79 , 11 ] . finally , the sample size and the age range of our subjects limit the generalization of the findings . within the above - mentioned limitations , it is concluded that the metabolic consequences of excessive body fat and low fitness are detectable early in life . taking into consideration the growing epidemic of childhood obesity , the public health authorities should seriously consider the design and early implementation of effective prevention strategies . health education programs aiming at the prevention of weight gain and the improvement of cardiorespiratory fitness in childhood could anticipate the metabolic abnormalities predisposed to or associated with mets risk and inflammation and thus contribute to the prevention of the mets and its complications later in life .

the aim of this study was to investigate the independent associations among cardiorespiratory fitness , metabolic syndrome ( mets ) , and c - reactive protein ( crp ) in children . the sample consisted of 112 children ( 11.4 0.4 years ) . data was obtained for children 's anthropometry , cardiorespiratory fitness , mets components , and crp levels . mets was defined using criteria analogous to the adult treatment panel iii definition . a mets risk score was also computed . prevalence of the mets was 5.4% , without gender differences . subjects with low fitness showed significantly higher mets risk ( p < 0.001 ) and crp ( p < 0.007 ) , compared to the high - fitness pupils . however , differences in mets risk , and crp between fitness groups decreased when adjusted for waist circumference . these data indicate that the mechanisms linking cardiorespiratory fitness , mets risk and inflammation in children are extensively affected by obesity . intervention strategies aiming at reducing obesity and improving cardiorespiratory fitness in childhood might contribute to the prevention of the mets in adulthood .

1. Introduction 2. Materials and Methods 3. Data Collection 4. Results 5. Discussion

PMC4379655

indirect aesthetic materials have become very popular in making of inlays , onlays , and crowns . lithium disilicate is one such material which has wonderful translucency and shade variety for the manufacture of inlays , onlays , and single crowns . indirect composite is another material which is proven to have improved fracture toughness because of post - cure treatment reinforcement . full - contour zirconia has become the most preferred material for anterior and posterior indirect restorations because of their high flexural strength , excellent biocompatibility , and good esthetics . limited data is available regarding the selection of the appropriate tooth - colored restorative material and its influence on the fracture resistance of cusp - replacing restorations in endodontically treated posterior teeth . the present study assessed the fracture resistance and failure patterns of endodontically treated maxillary premolars restored with three all - ceramic cad - cam ( computer aided design - computer aided manufacturing ) onlay materials which are lithium disilicate , indirect composite , and full zirconia . forty sound human maxillary premolars atraumatically extracted for orthodontic reasons were selected and were stored in 0.5% chloramine solution ( oxford lab chem , india ) . the roots were embedded in acrylic resin parallel to the long axis of teeth upto 2 mm below the cemento - enamel junction ( cej ) . ten teeth were randomly selected for the control group ( group i ) in which neither cavity preparation nor root canal treatment was performed . in the remaining 30 teeth , standardized mesio occluso distal ( mod ) onlay cavities were prepared using fg 271 and fg 169l carbide burs ( ss white , lake wood , usa ) . occlusal cavity width was kept at half the intercuspal distance , pulpal depth at 4 mm from the buccal cusp tip , and 6 taper on each wall . gingival seat in the proximal boxes were placed 1 mm above the cej and width of the seat in mesio - distal direction was 1.5 mm . the buccal and palatal cusps were reduced occlusally by 2 mm from cusp tips and a 1.5 mm width collar was prepared for both cusps . access openings were done with fg round diamond point iso 001 - 009 ( dia burs , mani , tochigi , japan ) . the coronal and middle thirds of the root canals were enlarged using sizes 1 - 3 gates glidden drills ( mani tochigi , japan ) . subsequently the instrumentation of the root canals was done by step back technique using hand k files till clean shavings of dentin was achieved . irrigation was performed using 1 ml of 2.5% naocl ( prime dental products , india ) after every change of file size and after cleaning and shaping 3 ml of 17% ethylenediaminetetraacetic acid ( edta ; prevest denpro , jammu and kashmir , india ) was irrigated for 1 minute to remove smear layer . the canals were obturated with gutta - percha ( densply maillefer , ballaigues , switzerland ) and grossman 's sealer ( pharma dent , montevideo , uruguay ) by lateral condensation technique . subsequently , the pulp chambers were temporarily filled with cavit ( 3 m espe , germany ) and stored for 24 h at 37c in 100% humidity . gutta - percha was removed upto the orifice of the canals and a zinc phosphate base of 1 mm was placed over the gutta - percha , and the remaining access cavity space was restored with dual cure flowable composite ( rebilda dc dual cure flowable composite , voco , germany ) till the pulpal floor . ii- indirect composite ( paradigm mz 100,3 m espe , st paul , mn ) and group iii- lithium disilicate glass - ceramic ( ips e. max cad , ivoclar vivadent , schaan , liechtenstein ) were fabricated using cad / cam ( cerec ; sirona , bensheim , germany ) with the cerec 3d system using the software package provided ( version 2.80r2402 ; sirona ) . in group iv- partially sintered zirconia onlays ( sagemax - nexxzr , federal way , usa ) were fabricated using cad / cam using delcam dent cad and roland milling machine . cementation surfaces of group iii onlays were etched using 5% hydrofluoric acid for 60 s and rinsed under running water for 60 s and air dried for 30 s. a silane coupling agent was applied and allowed to dry for 3 min . enamel and dentin margins were etched using 35% phosphoric acid for 15 s and rinsed with water for 30 s. all the onlays were cemented using multilink automix ( ivoclar vivadent , schaan , lichtenstein ) . multilink primer a and b were mixed and applied to the cavity preparations according to the manufacturer 's recommendation and the onlays were cemented and light - cured for 40 s on all restored surfaces . the same protocol was followed for cementation of groups ii and iv except 5% hydrofluoric etching step was eliminated , instead was replaced by air borne particle abrasion with 27 m aluminium oxide at 30 psi . the silane coupling agent ( metal / zirconia primer ) was applied for 180 s. after cementation of the restorations , specimens were stored in water for 21 days at 37c . the fracture resistance of the buccal cusp was tested on a universal testing machine ( instron , uk ) with load applied on the center of the buccal cusp slope with a steel ball of diameter 3.5 mm at an angle of 30 to the long axis of the tooth with a crosshead speed of 0.5 mm / min . after mechanical failure , all specimens were observed under stereomicroscope ( 2 magnification ) to visualize the fracture lines in the restoration and the tooth . failure patterns were evaluated and classified into five categories : type i : no visible fracture in the restoration and tooth type ii : no visible fracture in the restoration but fracture in the tooth type iii : fracture in the restoration only type iv : fracture in the restoration and the tooth above cej type v : fracture in the restoration and the tooth below cej resistance to fracture was submitted to parametric one - way analysis of variance ( anova ) test . these were performed using statistical package for social sciences ( spss ) , version # 16 . resistance to fracture was submitted to parametric one - way analysis of variance ( anova ) test . these were performed using statistical package for social sciences ( spss ) , version # 16 . the mean fracture resistance and standard deviation of the experimental groups is shown in figure 1 . groups ii and iii showed significantly lower values than the group i. however , groups ii and iii presented no significant difference from each other . group iii demonstrated 30% type ii fractures , 10% type iv , and 60% type v. group iv demonstrated 90% type i and 10% type ii fractures . bar graph representing mean fracture resistance of four groups bar graph representing comparison of failure patterns frequency table of failure patterns observed in all groups generally , endodontically treated teeth are considered to be more susceptible to fracture than the sound teeth , because of the internal tooth structure removal during endodontic treatment . according to reeh et al . , endodontic treatment performed in intact teeth reduced fracture resistance by 5% , but when combined with an mod cavity preparation , it is reduced to 69% . many authors preferred the onlay design to the inlay design as it would reinforce the cusps . in the present study , the bucco lingual width of half the intercuspal distance was maintained to simulate the clinical situation where a wide bucco lingual access cavity is prepared . earlier studies had maintained 1.5 - 2 mm of occlusal cavity width for the inlays and onlays . buccal and palatal cusps were reduced by 2 mm occlusally and are in line with bruke et al . , who suggested that 2 mm should be the minimal thickness of the indirect restoration that will provide adequate resistance to occlusal forces . in the present study , the fracture resistance of group i presented a mean fracture resistance of 980 n. similar values were found in other studies , which ranged between mean of 882 and 1,568n . this variance of results of the present study from the other studies may be due to difference in the type and design of the load application of the contact device , specimen preparation , test speed , and tooth storage method . studies were mainly done on indirect composite crowns and veneers assessing the fatigue resistance . in the present study , indirect composite cad onlays could not reinforce the endodontically treated teeth to that of sound teeth . again there are no studies in the literature assessing fracture resistance of e - max press and e - max cad onlays . however in the present study , e - max cad onlays could not reinforce the endodontically treated teeth to that of sound teeth . the mean fracture resistance in group iv was 1,524.4n which is highest of all the groups . this high fracture resistance of zirconia could be related to its phase transformation toughening phenomenon . under stresses , when crack propagates within the zirconia mass , the tetragonal grains are transformed to monoclinic with a volume expansion of 3 - 5% . this expansion of the grains will ultimately lead to compressive stresses at the edge of the induced crack front and so extra energy is required for the crack to propagate . in the present study , zirconia mod onlays had reinforced the endodontically treated to a much higher value than that of the sound teeth . in comparison between groups in the present study , the mean fracture resistance of group i was significantly higher than groups ii and iii . although the fracture resistance of groups ii and iii were lower than that of the group i , their fracture resistance values were much higher than the normal masticatory forces which is between 222 and 447 n. but that is not good enough to withstand the highest masticatory forces of 900n in bruxism and hard objects chewing . while comparing groups ii and iii , there is no significant difference statistically . this could be attributed to the increased thickness of e - max cad of 2 mm in this study which could have withstood the testing loads compared to ultrathin models in other studies . interestingly there is almost double the increase in the fracture resistance as compared to all the other groups . these findings were supported by ma et al . , who reported that flexural strength or fracture load of zirconia is 2.5 times greater than lithium disilicate glass ceramics suggesting that zirconia is much more suitable for free standing stress bearing application . coming to modes of failure ; in contrast to many studies , there was not much difference in the fracture modes of groups ii and iii even though the stress distribution varies with the material properties and cavity design . the occlusal cavity depth of 4 mm in the present study could have altered the stress distribution . this can be supported by goel et al . , where they suggested that deeper the prepared cavity , the greater the changes in the stress gradient in the dentin . the changes in the stress gradient could initiate fracture of the remaining cusps , possibly starting in the dentin immediately adjacent to the deepest portion of the cavity preparation . in group iv , none of the onlays fractured , but one sample fractured only in the root . the failure at a higher load could be due to the bond failure in the cement or fractures in the internal surface of the tooth , where more amount of stresses are usually concentrated . within the limitations of this in vitro study , it is interesting to note that full contour zirconia onlays had showed an excellent fracture resistance with no cracks in the restoration at very high loads , compared to lithium disilicate and indirect composite onlays . therefore , full contoured zirconia could be chosen as an ideal esthetic posterior onlay material and could be a good alternative to unesthetic metal onlays .

aim : to compare the fracture resistance and modes of failures of three different aesthetic mod onlays on endodontically treated premolars.materials and methods : forty sound maxillary premolars were selected of which 10 untreated teeth were taken as control ( group i ) . the other thirty premolars were subjected to standardized mod onlay preparations and root canal treatments and divided into 3 equal groups . onlays were prepared in group ii- indirect composite , group iii- lithium disilicate ceramic and group iv- full zirconia . all onlays were cemented using multilink automix . all the 40 samples were subjected to fracture resistance testing on universal testing machine . also fractured specimens were observed under stereo - microscope for modes of failure.results:group iv presented the highest fracture resistance . groups ii and iii presented no significant difference in fracture resistance from each other ( p > 0.05 ) . group ii and group iii showed significantly lower fracture resistance values than group i. coming to modes of failure , only group iv had showed no cracks in any of the restorations.conclusion:full zirconia mod onlays increased the fracture resistance of endodontically treated premolars to a significantly higher level than the sound teeth .

INTRODUCTION MATERIALS AND METHODS Statistical analysis RESULTS DISCUSSION CONCLUSION

PMC3306946

during the past few decades , dengue fever has gradually become one of the leading causes of morbidity and mortality in tropical and subtropical areas throughout the world . the dengue virus ( denv ) , a mosquito - borne member of the family flaviviridae , circulates as four distinct serological types denv 1 , denv 2 , denv 3 , and denv 4 . over all , two - fifth of the world population are living in areas , at risk for dengue [ 24 ] . infection with any of these leads to a mild self - limiting febrile illness ( dengue fever , df ) . a more severe form of the disease , dengue hemorrhagic fever / dengue shock syndrome ( dhf / dss ) , is responsible for high mortality rate , especially in children . it has been estimated that about 50 million cases of df occur annually , with 10,000 infant deaths due to dhf / dss . dhf / dss has been postulated to result from immune enhancement after a second heterologous denv infection . in india , denv was first isolated in 1946 and many outbreaks have been reported [ 710 ] . dhf was first reported in calcutta ( kolkata ) , west bengal in 1963 , again in 1964 . since then , there are numerous studies from the indian subcontinent investigating dhf in various parts of the country [ 1322 ] . but there are no studies investigating the overall prevalence of the dengue serotype circulating in the endemic zone , apart from the epidemic outbreak . the purpose of this paper is to present a comprehensive report on the diagnosis of dengue infection amongst the febrile cases , available from january to december , 2010 , in the city of kolkata and also to identify the serotype presently circulating in this region . the incidence of df in the rural west bengal is on the increase and is spreading to geographic regions not previously affected [ 23 , 24 ] . antibodies against group b - arthropod - borne viruses in more than 80% of the kolkata population have been recorded almost fifty years back , and that is too possibly due to the infection by dengue viruses . the present study aimed to identify the serotypes of denv in the population of kolkata as well as to study the sociodemographic status in relation to denv infection . the present population of kolkata is 44,86,679 of which 23,62,662 are males and 21,24,017 are females . the city has an international sea and airport and one rail way station ( sealdah ) which is busiest in the world . the rail road of this station covers a number of districts , situated at the border of bangladesh . adjacent to the city , there is a thickly populated town , howrah , which has one of the biggest terminating railway stations of the eastern india . cases were mainly referred from outpatient department ( opd ) and indoor of i.d & b. g hospital , attached to this unit , from different medical colleges as well as other hospitals in kolkata along with a short history of the patients . a good number of cases were referred to us by the private practitioners also . in the matter of selection of dengue fever ( df ) cases , the following criteria were initially considered : ( 1 ) high fever ; ( 2 ) head ache ; ( 3 ) retro - orbital pain ; ( 4 ) nausea / vomiting ; ( 5 ) malaise / joint pain ; ( 6 ) generalized skin rashes . in the present study two or more of these criteria , apart from fever , were fulfilled . the possibilities of bacterial and prokaryotic etiology in the collected samples were excluded through investigations at the respective hospitals . the case history and the investigations of the patients were compiled . in the case of dhf , the history of illness was revealed by the sudden rise of high fever ( 38.3c39.4c ) , headache , retro - orbital pain , conjunctival congestion , and facial flashing . in addition to that , some cases had the history of hemorrhagic manifestation either with petechiae or with gum bleeding or malena . in such cases , intermittent or biphasic course of fever was recorded , where the first phase of fever persisted for 27 days and the second bout of fever persisted for 23 days . no cases with the history of plasma leakage were observed . in the admitted cases , we found that fever was also accompanied by generalized malaise and lumbosacral pain . those patients represented rashes , appeared on 25 days of illness in limbs , sparing palm , and souls . sera were separated from the collected blood samples , were stored at 80c , and were tested within 1 month from the date of collection . a total of 378 samples were thus received from the suspected cases and analyzed for the detection of dengue viruses , if any . to study the sociodemographic status in relation to denv infection , all the 378 samples were screened for the presence of dengue igm antibodies by igm capture enzyme - linked immunosorbent assay ( elisa ) : using a kit , prepared by the national institute of virology , pune , india , following the prescribed protocol . optical density ( od ) was measured at 492 nm using an elisa reader ( titertek multiskan plus , lab systems finland , type-314 ) . to study the molecular typing of denv , attempts were made to isolate the rna from all the samples as well as from four different denv serotype strains , which were used as positive control . viral rna was isolated by using qiagen viral rna isolation kit ( qiagen , gmbh , hilden , germany ) according to the manufacturer 's protocol . in this study , , viral rna was converted to a dna copy ( cdna ) prior to enzymatic dna amplification by the use of reverse transcriptase ( rt ) and the denv downstream consensus primer d2 - 5-ttgcaccaacagtcaatgtcttcaggttc-3 homologous to the genomic rna of the four serotypes . subsequent taq polymerase amplification was performed on the resulting cdna with the upstream dengue virus consensus primer d1 - 5-tcaatatgctgaaacgcgcgagaaaccg-3. target rna was amplified in 25 l volumes containing the following components : 800 mm deoxynucleotide triphosphates ( dntps ) , 8 mm dithiothreitol , 0.24 m each of primers d1 and d2 , 0.5 u of amv rt ( promega , madison , wi , usa ) , and 0.625 u of dreamtaq dna polymerase ( fermentas inc . , the reactions were allowed to proceed for 1 h at 42c and then to proceed with 95c for 3 minutes for initial denaturation followed by 35 cycles of denaturation ( 95c for 30 sec ) , primer annealing ( 55c for 1 min ) , and primer extension ( 72c for 2 min ) along with final extension ( 72c for 5 min ) . denv serotyping was conducted by second - round amplification ( nested pcr ) initiated with 10 u of diluted material ( 1 : 100 in sterile distilled water ) from the initial amplification reaction . the total 20 l of reaction mixture was prepared using 2 l of diluted first pcr products , 0.8 mm dntps , 0.5 u of dreamtaq dna polymerase and 0.3 m of primer d1 and 0.3 m of dengue virus type - specific primers : ts1 5-cgtctcagtgatccggggg-3 , ts2 5-cgccacaagggccatgaacag-3 , ts3 5-taacatcatcatgagacagagc-3 , and ts4 5-ctctgttgtcttaaacaagaga-3. dithiothreitol and amv rt were eliminated . the samples were subjected to initial denaturation ( 95c for 3 min ) followed by 20 cycles of denaturation ( 95c for 30 s ) , primer annealing ( 55c for 1 min ) , and primer extension ( 72c for 1 min ) along with final extension ( 72c for 5 min ) . the pcr products were analyzed by running a 1.5% agarose gel stained with ethidium bromide . out of 378 samples collected , only 173 samples were reactive to dengue igm antibody by elisa method . maximum numbers of igm positive cases were observed in the age group of 010 years in both male and female patients ( figure 1 ) . females were more affected ( 46.5% ) than the males ( 45.1% ) . as regards the seasonal prevalence , it is evident from the result that although sporadic cases obtained throughout the year , dengue cases started from the month of july and attained maximum number of cases in the month of november ( figure 2 ) . out of 378 samples , seventy - four samples ( 68.5% ) had the denv infection by single different serotypes , of which 7 samples ( 9.5% ) had the monotypic infection with denv-1 , 45 samples ( 60.8% ) had the monotypic infection with denv-2 and 22 samples ( 29.7% ) had the monotypic infections with denv-3 serotype . only one sample had all the three serotypes , that is , denv-1 , denv-2 , and denv-3 . out of 22 dhf cases , 3 were found with single - serotype infection and 18 cases with dual infections of denv 2 and denv 3 . only a female patient of 24 years of age had the infection with denv-1 , denv-2 , and denv-3 serotypes at a time and also suffered from dhf . clinical data regarding the death of the dhf cases was inadequate to reach any conclusion about the severity of illness . the specimen containing denv-1 , denv-2 , and/or denv-3 is identified by the detection of a dna band of 482 , 119 , or 290 bp in size , respectively , on 1.5% agarose gel , loaded with nested pcr products along with positive controls , stained with ethidium bromide ( figure 3 ) . the monitoring of denv activity is required for public health importance , as the dengue fever and dhf / dss are increasing worldwide and are spreading in the places , where it was previously not reported . the first isolation of denv serotype 1 and 4 was reported from india in 1964 [ 31 , 32 ] and serotype 3 in 1968 . although concurrent infection with more than one serotype of denv in the same individual is uncommon , high percentage of concurrent infections with different denv serotypes had been detected at an outbreak in delhi , india , in 2006 . kolkata is a dengue endemic zone ; frequent outbreaks of df and dhf have been occurring since last centuries . in kolkata , dengue was first documented in 1824 and several epidemics took place in the city during the years 1836 , 1906 , 1911 , and 1972 , affecting 40% of the city people . the last large - scale dengue outbreak has been recorded in the year 2005 . in the rural areas of west bengal , dengue is gradually spreading and establishes new reports [ 23 , 24 ] . no continuous monitoring of the molecular detection of the dengue serotype has yet been attempted in the city of kolkata , either in epidemic or in sporadic dengue outbreaks . out of 378 blood samples , agewise distribution revealed that the highest number of dengue cases were detected in the age group of 010 years , followed by 1120 years and above ( figure 1 ) . in the highest age group ( above 50 years ) , the number of positive cases were too small and only 16.6% dengue igm positivity was found in the male individuals . in all the age groups , females were more affected than the males ( figure 1 ) . are domestic and peridomestics in nature , the females get more exposure than the males , as most of the time they reside inside the house . during the year - round study , although small number of samples from suspected dengue cases were referred to us during the period from january to may , only a few were positive to dengue igm antibody by elisa method . it is evident from our study ( figure 2 ) that the dengue cases actually started from the month of june and attained its peak in the month of november during this year , which is the post - monsoon period . it may be explained by the fact that the stagnant fresh water during the rainy seasons ( june to october ) favoured the breeding of the vector mosquitoes . for the molecular detection , rt - pcr was performed with all samples , of which 108 samples were positive by that method . a total of 74 samples had the monotypic infections , involving denv 1 , denv 2 , and denv 3 , of which denv 2 was predominated . conversely , 33 samples had the dual infections with denv 2 and denv 3 , of which all the samples were igm reactive . only one sample produced prominent band against denv1 , denv 2 , and denv 3 and also contained igm antibody . the detection of the viral rna in presence of the igm antibody may be explained by the fact that , due to the consecutive infection , the igm detected in those samples possibly appeared due to the initial infection , which is evident by the intensity of the bands in gel electrophoresis ( figure 3 ) . the hemorrhagic manifestation was found mainly among the patients , infected with multiple dengue serotypes . dhf cases were observed mainly among the young and young - adult age groups ( 030 years ) which might be due to the absence of immunity against all serotypes of denv in them . although the increasing trend of cocirculation of multiple denv serotypes suggests that kolkata is becoming a hyperendemic state , a large - scale monitoring on the circulating strains of denv is highly required to draw a definite conclusion .

dengue is one of the major public health threats in kolkata . every year , blood samples with dengue - like illness are referred to us from different medical colleges and hospitals in kolkata for the detection of dengue infection in them . in 2010 , a total of 378 samples were referred to us for that purpose . all the samples were tested for the detection of igm antibodies by elisa method , followed by rt - pcr test for the detection of serotypes . only 173 samples were elisa positive . out of 378 samples , 108 were rt - pcr positive . out of 108 samples , 74 samples had monotypic infection with different serotypes of denv and 33 samples had dual infections with denv-2 and denv-3 . only one sample had the infection with denv-1 , denv-2 , and denv-3 . dhf was found mainly among the patients , infected with multiple dengue serotypes . only 3 dengue monotypic infected patients had suffered from dhf .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

PMC3809738

hypovitaminosis d is a highly prevalent condition , especially in older adults , with an estimated prevalence of 5090% depending on the definition used and the population studied [ 1 , 2 ] . growing evidence has established that vitamin d exhibits not only skeletal effects but also a number of nonskeletal effects [ 1 , 2 ] . this finding explains why vitamin d plays a much greater role in human health than previously thought and why lower 25-hydroxyvitamin d ( 25ohd ) concentrations contribute to cellular dysfunction in many biological systems , which in turn is manifested by organ disorders and chronic diseases [ 14 ] . in particular , it has been reported , among older inpatients hospitalized in geriatric acute care units , that lower serum 25ohd concentrations at the time of admission are directly associated with a greater severity of chronic diseases and higher risks of acute decompensation and inhospital mortality . in line with this , hypovitaminosis d has been proposed as a biomarker of longer length of stay ( los ) in acute care units [ 79 ] . for instance , lower serum 25ohd concentrations were independently associated with longer intensive care unit stay in cardiac surgical patients . hypovitaminosis d also doubled the risk of being hospitalized more than 14 days in a geriatric acute care unit and , in another study , the combination of hypovitaminosis d , male gender , and delirium predicted a 4.8-fold higher risk of longer los among geriatric inpatients . importantly , the average increase in los in geriatric acute care units attributable to hypovitaminosis d remains unknown . moreover , while existing studies report an association between hypovitaminosis d and prolonged hospitalization , it remains unclear whether an inverse linear association exists over a range of 25ohd concentrations and whether any increase in 25ohd is associated with a decrease in los , regardless of the initial level . we had the opportunity to examine these issues in a historical cohort of older inpatients hospitalized in a geriatric acute care unit . the objectives of this analysis were ( i ) to estimate the difference in hospitalization days between inpatients with and without hypovitaminosis d and ( ii ) to determine whether there was a linear association between serum 25ohd concentration and los . the studied sample was a convenience of all inpatients consecutively admitted into the geriatric acute care unit of angers university hospital , france , between june and december 2008 . study inclusion criteria included unplanned admissions to hospital for people 75 years and willingness to participate . two hundred fifty - three inpatients met the study inclusion criteria and agreed to participate . power analysis determined that 253 participants were sufficient to find significant correlations between serum 25ohd concentration and los with 77% power at p < 0.05 . the study was conducted in accordance with the ethical standards set forth in the helsinki declaration ( 1983 ) . the los was calculated in number of days using the administrative registry of angers university hospital by subtracting day of admission from day of discharge . the age , gender , usual place of life , degree of functional independence , main reason for admission , number of acute diseases and comorbidities , use of vitamin d supplements , and creatinine clearance were assessed as part of a standardized comprehensive geriatric assessment on admission to hospital and were used as potential confounders . functional independence was measured from 0 to 6 ( best ) with the autonomy , gerontology , iso - resources groups ( aggir ) model . the usual place of life was also sought ( i.e. , home living versus institution dwelling defined as living in nursing home or in senior housing facilities ) . the main reasons for admission were separated into 6 categories : gait disorders / falls / orthopedic reasons , cardiovascular disorders , respiratory disorders , digestive disorders , neuropsychiatric disorders , and social issues . acute diseases were defined as diseases with sudden onset and rapid progression , whatever their nature or site . comorbidities were diseases lasting at least 3 months or running a course with minimal change , whatever their nature or site . vitamin d supplementation was systematically noted from the primary care physician 's prescription and sought by questioning the patient , whatever the dosage schedule or route of administration and regardless of the date of commencement . fasting early morning venous blood was collected from resting participants on the first day of hospital stay ( i.e. , the same day as the standardized anamnesis and physical examination ) for the measurement of serum 25ohd and creatinine . serum 25ohd concentration was measured by radioimmunoassay ( diasorin inc . , stillwater , mn ) with intra- and interassay precisions of 5.2% and 11.3% , respectively . hypovitaminosis d was defined as serum 25ohd 50 nmol / l ( to convert to ng / ml , divide by 2.496 ) according to the definition of the world health organization and the institute of medicine . creatinine clearance was estimated using cockcroft - gault formula ( [ ( 140 ageyears ) weightkg / creatininemol / l ] 1.04 for females and 1.25 for males ) . participants ' characteristics were summarized using means and standard deviations or frequencies and percentages , as appropriate . as the number of observations was higher than 40 , comparisons were not affected by the shape of the error distribution and no transform was applied . first , comparisons between participants separated into two groups based on hypovitaminosis d were performed using chi - square test or student 's t - test , as appropriate . second , univariate and multiple ( i.e. , fully adjusted direct regression and backward regression ) linear regressions were used to examine the association between los in geriatric acute care unit ( dependent variable ) and serum 25ohd concentration ( independent variable ) , while adjusting for potential confounders . third , a pearson correlation was used to examine the relationship between serum 25ohd concentration and los in the unit . all statistics were performed using spss ( v19.0 , ibm corporation , chicago , il ) . among 253 inpatients included in this analysis ( mean age standard deviation86.2 6.0 years ; 66.8% female ; 100% caucasian ) , the mean serum 25ohd concentration was 33.9 21.1 nmol / l and the mean los was 14.7 8.1 days . we found that the los was longer among older inpatients with hypovitaminosis d compared to those with adequate vitamin d status ( 15.2 8.2 days versus 12.1 7.0 days , p = 0.017 ) , underlining a mean los difference of 3.1 days between groups ( table 1 ) . there were no other demographical and clinical differences , except for the use of vitamin d supplements ( 4.8% in participants with hypovitaminosis d versus 38.9% in their counterparts , p < 0.001 ) . univariate and multiple linear regression models showed an inverse association between serum 25ohd concentration and los ( unadjusted = 0.05 [ 95% confidence interval ( ci ) : 0.10 ; 0.01 ] , p = 0.028 ; = 0.07 [ 95% ci : 0.14 ; 0.02 ] , p = 0.043 for adjusted model ; = 0.08 [ 95% ci : 0.14 ; 0.02 ] , p = 0.015 for backward model ) . the creatinine clearance was also retained as an explanatory variable of los in the backward model ( = 0.02 [ 95% ci : 0.05 ; 0.01 ] , p = 0.081 ) . finally , as illustrated in figure 1 , we found an inverse correlation between serum 25ohd concentration and los ( r = 0.14 , p = 0.028 ) . our results show that , in a geriatric acute care unit , older inpatients with serum 25ohd 50 nmol / l at time of admission had a los extended by approximately 3 days compared to those with 25ohd > 50 nmol / l . additionally , we report the first evidence of a linear association between 25ohd concentration and los , suggesting that any increase in 25ohd was associated with a decrease in los whatever the initial vitamin d status . vitamin d is a secosteroid hormone that exhibits multiple biological actions mediated by the vitamin d receptor present in many cells and target tissues [ 13 ] . since vitamin d is necessary for the regulation of cellular growth , differentiation , and function , lower vitamin d status leads to multiple organ dysfunction , disability , and unstable health status , which are all causes of deconditioning , polypharmacy , and longer los [ 1416 ] . consistently , previous literature from our group has provided compelling evidence that hypovitaminosis d could be regarded as a biomarker of age - related disease burden [ 4 , 5 ] and consequent longer los [ 79 ] . for instance , it has been reported that the risk of being in the highest tertile of los was multiplied by approximately 2 in case of hypovitaminosis d at baseline among geriatric inpatients . however , the number of additional hospitalization days in case of hypovitaminosis d has not been examined yet . thus , the results of the current study provide extra information , specifically , that older inpatients with hypovitaminosis d were hospitalized 3 days more in our geriatric acute care unit than those with adequate vitamin d status ; that is , they had a 21 percent longer los ( table 1 ) . the clinical relevance is that longer los exposes older inpatients to greater risks of disability , nosocomial infections , deconditioning , and higher healthcare costs [ 16 , 17 ] . consequently , any decrease in los is beneficial to older inpatients . in the current study , we found a 0.14-day decrease in los per 1 nmol / l increase in serum 25ohd concentration ( figure 1 ) , that is , a 1-day decrease in los per 7.1 nmol / l increase in 25ohd . it has been previously reported that vitamin d supplementation produces an elevation of serum 25ohd concentration of approximately 2.5 it can thus be inferred that each additional 300 iu vitamin d per day is associated with a 1-day shortening of los among geriatric inpatients . such estimates may help to justify , plan , evaluate , and compare the effectiveness of interventions aiming at shortening the los in geriatric acute care units with vitamin d supplements firstly , the study cohort was restricted to caucasian older inpatients hospitalized in a single geriatric acute care unit who might be unrepresentative of all older inpatients . thirdly , although we were able to control for many important characteristics that could bias the results , residual prehospital health conditions influencing both vitamin d status and los , such as the body mass index , were not considered . finally , it has to be kept in mind that , in some cases , los does not only depend on bioclinical aspects but is influenced by other factors , including number of beds available , human resource employees , and health insurance guidelines , which could not be taken into account here . increased serum 25ohd concentrations were linearly associated with shorter los in this cohort of acute care geriatric inpatients . those with an adequate vitamin d status were hospitalized 3 days less than those with hypovitaminosis d. our findings reinforce the conceptualization of vitamin d as a biomarker of the health status of inpatients in geriatric acute care unit and provide an additional rationale for prescribing vitamin d supplements in this population . multicenter randomized controlled trials are required to investigate the benefits of vitamin d supplementation on los with higher levels of evidence .

background . hypovitaminosis d is linked to unstable health in older adults . our objectives were to determine ( i ) the difference in length of stay ( los ) in geriatric acute care unit between inpatients with and without hypovitaminosis d and ( ii ) whether there was a linear association between serum 25-hydroxyvitamin d ( 25ohd ) concentration and los . methods . 253 inpatients admitted in 2008 to the geriatric acute care unit of angers university hospital , france , ( mean agestandard deviation , 86.2 6.0 years ; 66.8% female ) were included in this historical cohort study . los was calculated by subtracting day of admission from day of discharge . hypovitaminosis d was defined as 25ohd50 nmol / l at the time of admission . age , gender , place of life , functional independence , reason for admission , number of acute diseases and comorbidities , use of vitamin d supplements , and creatinine clearance were used as confounders . results . participants with hypovitaminosis d had longer los than their counterparts ( 15.2 8.2 days versus 12.1 7.0 days , p = 0.017 ) , underlining a mean difference of 3 days . 25ohd concentration inversely correlated ( r = 0.14 , p = 0.028 ) and was inversely associated with los ( adjusted = 0.07 [ 95%ci : 0.14 ; 0.02 ] , p = 0.043 ) . conclusions . we found an inverse linear association between serum 25ohd concentrations and los in a geriatric acute care unit . participants with 25ohd>50 nmol / l were hospitalized on average 3 days less than those with 25ohd50 nmol / l .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

PMC3059852

body height was associated with race time in both male and female marathon runners,8 body mass index was related to marathon running time in females,10 and body fat was positively associated with marathon race times in females.10 the relationship between selected skin - fold thicknesses and running performance has been investigated in several studies . hagan et al . demonstrated that apart from other variables , the sum of skin - fold thicknesses was correlated to marathon race time in males.20 bale et al . reported the sum of skin - fold thicknesses , the type and frequency of training and the number of years running were the best predictor variables for 10-km race time in males.6 in recent studies , a relationship between the thicknesses of selected skin - folds and running performance has been demonstrated for high - level runners.13,14 in these studies , elite male and female runners of distances from 100 m to 10 km and the marathon had been investigated.13,14 high correlations were found between the front thigh and medial calf skin - fold and 10-km race times for male runners , and between iliac crest and abdominal skin - fold and marathon times for female runners.13 it was supposed that the reduced thickness of skin - folds of the lower limb were a result of high intensity in running training.14 legaz & eston concluded from their study of high - level runners that running training led to a decrease in the sum skin - folds and the skin - fold thickness at the abdominal , front thigh and medial calf sites.14 further , the lower limb skin - fold thicknesses might be a useful predictor variable of running performance.13,14 apart from anthropometry , volume and intensity in training also seemed to influence running performance in long - distance runners up to the marathon distance . regarding volume , in marathon finishers , the longest distance ran per training unit was the best predictor variable for marathon race time.21 male runners completing more than 100 km per week had significantly faster race times over 10 to 90 km than athletes covering less than 100 km22 and elite runners with a higher training frequency , higher weekly training volume and longer running experience competed faster in a 10 km run.6 in female marathoners , the number of weekly training sessions and the number of years training were the best predictors for race time.23 in long - distance runners , training units of moderate intensity were related to race performance.24 in female marathoners , a faster marathon time was associated with higher aerobic capacity and years of training rather than with body dimensions.25 male and female top class marathon runners trained for more total kilometres per week and at a higher velocity than runners at a lower level,26 and peak running velocity during training was highly related to 5 km run times for both male and female athletes.27 when training in runners was analysed in details , several parameters such as training days , total training sessions , total kilometres , mean kilometres per training session , longest mileage covered per training session , total training minutes , maximal kilometres ran per week , mean kilometres per week and mean kilometres per day , were related to marathon race time.10,20,21 what this background shows , is that the relationship between skin - fold thicknesses and running performance has been investigated in running distances from 100 m to the marathon distance , except the half marathon distance.13,14 the intention was therefore to investigate whether a relationship exists between selected anthropometric variables including skin - fold thicknesses and training variables with half marathon race performance in recreational female runners . it was hypothesised that significant relationships would be found between upper body skin - fold thicknesses ( suprailiacal and abdominal site ) and half marathon race times for recreational female runners . we also intended to create an equation to predict a half marathon race time for future novice female half marathoners , based upon basic measurements any athlete could determine for himself without the need for highly sophisticated equipment . we performed a cross - sectional observational research at a half marathon race during the half marathon basel in switzerland . the organiser contacted all participants of the half marathon basel in 2010 via a separate newsletter , three months before the race , in which they were asked to participate in the study . half marathon basel took place on 12 september 2010 , in the city of basel , switzerland . the athletes started at 11:00 a.m. and had to run one flat lap on asphalt . the temperature was 13 celsius at the start and the relative humidity was at 63% . a total of 396 female athletes started in the half marathon ; 42 female runners were interested in participating in our investigation . half marathon basel within the time limit of 2:30 h : min . before the start of the race body mass , body height , and thicknesses of skin - folds were measured . with this data we calculated body mass index and percent body fat using an anthropometric method . body mass was measured to the nearest 0.1 kg using a beurer bf15 scale ( beurer gmbh , ulm , germany ) . body mass index ( kg / m ) was calculated from body mass and body height . skin - fold thicknesses were measured by the same investigator at the following eight sites : pectoral , mid - axilla , triceps , subscapular , abdominal , suprailiac , front thigh and medial calf . skin - fold data was obtained using a skin - fold calliper ( gpm - hautfaltenmessgert , siber & hegner , zurich , switzerland ) and recorded to the nearest 0.2 mm . the measurements were made three times on the right side and the mean of the three measurements was used for the analyses . the timing of the taking of the skin - fold measurements was standardised to ensure reliability . one trained investigator took all the skin fold measurements as intertester variability is a major source of error in skin - fold measurements . an intratester reliability check was conducted on 27 male and 11 female runners prior to testing . intra - class correlation ( icc ) within the two judges was excellent for both men and women for all anatomical measurement sites ( icc>0.9).28 readings were performed 4 s after applying the calliper , according to becque et al.29 percent body fat was calculated using the formula : percent body fat = -6.40665 + 0.41946 ( 3sf ) 0.00126 ( 3sf ) + 0.12515 ( hip ) + 0.06473 ( age ) using the formula of ball et al.30 3sf was taken as the sum of the three skin - fold thickness of the triceps , suprailiac and front thigh skin - fold thicknesses . volunteers were asked to maintain a comprehensive training diary during the 3-month period before the race . the training records consisted of the number of training units with duration , kilometres and pace , weekly kilometres ran , weekly hours ran , and minimal and maximal kilometres ran per week . the athletes recorded their running speed during training in min / km and reported on the number of years that they had actively participated in running . the coefficient of variation of performance ( cv% = 100 sd / mean ) for total race time was calculated . in a first step , the association of the variables of anthropometry , training and pre race experience with total race time was investigated using bivariate correlation analysis . given the multiple tests , bonferroni correction was applied for n = 22 variables ( p = 0.0022 ) . in a second step , multiple linear regression analysis was used to further investigate the relationship of variables with significance in the bivariate analysis to race time . a probability value of less than 0.05 was accepted as significant for the multiple linear regression analysis . the half marathon basel took place on 12 september 2010 , in the city of basel , switzerland . the athletes started at 11:00 a.m. and had to run one flat lap on asphalt . the temperature was 13 celsius at the start and the relative humidity was at 63% . a total of 396 female athletes started in the half marathon ; 42 female runners were interested in participating in our investigation . before the start of the race body mass , body height , and thicknesses of skin - folds were measured . with this data we calculated body mass index and percent body fat using an anthropometric method . body mass was measured to the nearest 0.1 kg using a beurer bf15 scale ( beurer gmbh , ulm , germany ) . body mass index ( kg / m ) was calculated from body mass and body height . skin - fold thicknesses were measured by the same investigator at the following eight sites : pectoral , mid - axilla , triceps , subscapular , abdominal , suprailiac , front thigh and medial calf . skin - fold data was obtained using a skin - fold calliper ( gpm - hautfaltenmessgert , siber & hegner , zurich , switzerland ) and recorded to the nearest 0.2 mm . the measurements were made three times on the right side and the mean of the three measurements was used for the analyses . the timing of the taking of the skin - fold measurements was standardised to ensure reliability . one trained investigator took all the skin fold measurements as intertester variability is a major source of error in skin - fold measurements . an intratester reliability check was conducted on 27 male and 11 female runners prior to testing . intra - class correlation ( icc ) within the two judges was excellent for both men and women for all anatomical measurement sites ( icc>0.9).28 readings were performed 4 s after applying the calliper , according to becque et al.29 percent body fat was calculated using the formula : percent body fat = -6.40665 + 0.41946 ( 3sf ) 0.00126 ( 3sf ) + 0.12515 ( hip ) + 0.06473 ( age ) using the formula of ball et al.30 3sf was taken as the sum of the three skin - fold thickness of the triceps , suprailiac and front thigh skin - fold thicknesses . volunteers were asked to maintain a comprehensive training diary during the 3-month period before the race . the training records consisted of the number of training units with duration , kilometres and pace , weekly kilometres ran , weekly hours ran , and minimal and maximal kilometres ran per week . the athletes recorded their running speed during training in min / km and reported on the number of years that they had actively participated in running . normally distributed data are presented as mean and standard deviation ( sd ) . the coefficient of variation of performance ( cv% = 100 sd / mean ) for total race time was calculated . in a first step , the association of the variables of anthropometry , training and pre race experience with total race time was investigated using bivariate correlation analysis . given the multiple tests , bonferroni correction was applied for n = 22 variables ( p = 0.0022 ) . in a second step , multiple linear regression analysis was used to further investigate the relationship of variables with significance in the bivariate analysis to race time . a probability value of less than 0.05 was accepted as significant for the multiple linear regression analysis . the 42 athletes finished the half marathon basel within 119 ( 15 ) min ( cv = 12.6% ) , running at a mean speed of 10.8 ( 1.4 ) km / h . for the anthropometric characteristics , body mass , body mass index and body fat percentage ( see table 1 ) as well as pectoral , mid - axilla , triceps , subscapular , abdominal , suprailiac , and medial calf skin - folds ( see table 2 ) correlated to race time in the bivariate analysis . for the training characteristics , the mean speed of the training sessions was highly significantly and positively related to race time ( see table 3 ) . when the variables with significant association in the bivariate analysis were inserted into a linear regression model ( see table 4 ) , mid - axilla skin - fold and mean speed of the training sessions were related to race time . race time in a half marathon might be predicted by the following equation ( r = 0.71 ) for recreational female runners : race time ( min ) = 166.7 + 1.7x ( mid - axilla skin - fold thickness , mm ) - 6.4x ( speed in training , km / h ) . mean running speed during training was related to mid - axilla , subscapular , abdominal , and suprailiacal skin - fold thicknesses , the sum of eight skin - folds and percent body fat ( see table 5 ) . the aim of this study was to find predictor variables for half marathon race time in recreational female runners in order to create an equation to predict race time for novice future runners . we hypothesised that , according to the existing literature of arrese & ostriz , significant relationships will be found between the upper body skin - fold thicknesses at suprailiacal and abdominal site and the half marathon race times of recreational female runners.13 as we hypothesised , an association between upper body skin - fold thicknesses and race performance was found in the bivariate analysis ; pectoral , mid - axilla , triceps , subscapular , abdominal and suprailiac skin - folds were related to half - marathon running times . arrese & ostriz described in their 11 female high - level marathon runners a significant relationship between iliac crest ( r = 0.62 , p = 0.042 ) and abdominal skin - fold thickness ( r = 0.61 , p = 0.046 ) with marathon race times.13 we can confirm their findings and in addition , we also found for pectoral , mid - axilla , triceps , and subscapular sites significant associations . the female top class marathoners of arrese & ostriz with a body mass of 45.6 kg and a body height of 1.58 m had a body mass index of 18.3 kg / m compared to the 21.2 kg / m of our subjects.13 we must assume that our recreational females with a higher body mass index also had a higher body fat percentage , and consequently thicker skin - folds compared to the high - level marathoners of arrese & ostriz.13 in the bivariate analysis , mean speed of the training sessions was highly significantly and negatively correlated to half marathon race times . these findings confirm recent findings where peak running velocity during training was highly related to 5 km run times for both male and female athletes.27 according to hagan et al . , however , both variables of anthropometry and training seem to account for marathon race time in females.10 in their sample of 35 female distance runners , marathon performance time was related to body mass index , maximal oxygen uptake , previous marathons completed , number of weekly training sessions , training session per two days , total number of training sessions , total training duration , training pace and distance in training . we can confirm their findings that both variables of anthropometry and training were associated with half marathon running times in our subjects after multivariate analysis and not only anthropometric or training variables . based upon previous studies of runners up to the marathon distance , we expected to find an association between lower body skin - fold thicknesses and variables of training . legaz & eston described in a sample of 24 male and female endurance runners a significant association between the decrease in front thigh skin - fold thickness and improvement in performance due to training ( r = -0.74 , p<0.001).14 we found , however , an association of mid - axilla , subscapular , abdominal and suprailiac skin - fold thickness with running speed during training . legaz & eston investigated 16 male and eight female spanish high - level runners covering distances between 100 m and the marathon.14 their athletes trained six or seven days a week , for 20 to 25 hours . in contrast , our recreational runners trained for only three hours a week . also in the study of arrese & ostriz , where iliac crest and abdominal skin - fold thicknesses were related to marathon performance in females , high - level runners had been investigated.13 furthermore we must assume that our runners were older and had a higher body fat percentage compared to the elite runners in legaz & eston14 and arrese & ostriz.13 the subjects in the study of arrese & ostriz were between 21 years ( 100 m ) and 30 years ( marathon).13 the mean age of our subjects was 38 years , and we might expect that older runners would have more body fat . a cross - sectional study is limited regarding the influence and effects of anthropometric and training characteristics on race time in runners , since only an intervention trial can answer this question . unfortunately we have no data about energy deficit31 or disorder in fluid or electrolyte metabolism32 which also might affect performance . however , in existing literature , smaller samples were investigated . in the study of arrese & ostriz,13 11 female marathoners were included and legaz & eston14 investigated a total of eight female endurance runners . a cross - sectional study is limited regarding the influence and effects of anthropometric and training characteristics on race time in runners , since only an intervention trial can answer this question . unfortunately we have no data about energy deficit31 or disorder in fluid or electrolyte metabolism32 which also might affect performance . however , in existing literature , smaller samples were investigated . in the study of arrese & ostriz,13 11 female marathoners were included and legaz & eston14 investigated a total of eight female endurance runners . to summarize , both variables of anthropometry and training were related to half marathon race time in these recreational female runners and an association between upper body skin - fold thicknesses and speed in running during training was found . intensity in training seemed to be of importance for a fast half marathon race time and high training intensity may lead to reduced thickness of upper body skin - folds in recreational female half marathoners .

introduction : the relationship between skin - fold thickness and running performance has been investigated from 100 m to the marathon distance , except the half marathon distance.objective:to investigate whether anthropometry characteristics or training practices were related to race time in 42 recreational female half marathoners to determine the predictor variables of half - marathon race time and to inform future novice female half marathoners.methods:observational field study at the half marathon basel in switzerland.results:in the bivariate analysis , body mass ( r = 0.60 ) , body mass index ( r = 0.48 ) , body fat ( r = 0.56 ) , skin - fold at pectoral ( r = 0.61 ) , mid - axilla ( r = 0.69 ) , triceps ( r = 0.49 ) , subscapular ( r = 0.61 ) , abdominal ( r = 0.59 ) , suprailiac ( r = 0.55 ) medial calf ( r = 0.53 ) site , and speed of the training sessions ( r = -0.68 ) correlated to race time . mid - axilla skin - fold ( p = 0.04 ) and speed of the training sessions ( p = 0.0001 ) remained significant after multi - variate analysis . race time in a half marathon might be predicted by the following equation ( r2 = 0.71 ) : race time ( min ) = 166.7 + 1.7x ( mid - axilla skin - fold , mm ) - 6.4x ( speed in training , km / h ) . running speed during training was related to skin - fold thickness at mid - axilla ( r = -0.31 ) , subscapular ( r = -0.38 ) , abdominal ( r = -0.44 ) , suprailiacal ( r = -0.41 ) , the sum of eight skin - folds ( r = -0.36 ) and percent body fat ( r = -0.31).conclusion : anthropometric and training variables were related to half - marathon race time in recreational female runners . skin - fold thicknesses at various upper body locations were related to training intensity . high running speed in training appears to be important for fast half - marathon race times and may reduce upper body skin - fold thicknesses in recreational female half marathoners .

INTRODUCTION MATERIALS AND METHODS The Race Measurements and Calculations Statistical Analysis RESULTS DISCUSSION Limitations of the study CONCLUSION

PMC3667644

you need : a vu - ams5fs ambulatory recording device ( including an infrared interface cable that either connects to the rs232 serial port of a pc or to a usb port).7 electrodes ( we used conmed 1690 - 003).2 charged aa-batteries.an empty compactflash memory card ( the vu - ams5fs has been extensively tested with the 1 gb 80x cf card from transcend ( ts1gcf80 ) , but other cf cards should work too).a laptop or pc with flash card reader and the data analysis management software ( dams ) suite installed.a stopwatch.music player with children 's stories and headphones and a small self - inflatable air mattress are optional . a vu - ams5fs ambulatory recording device ( including an infrared interface cable that either connects to the rs232 serial port of a pc or to a usb port ) . 7 electrodes ( we used conmed 1690 - 003 ) . 2 charged aa - batteries . an empty compactflash memory card ( the vu - ams5fs has been extensively tested with the 1 gb 80x cf card from transcend ( ts1gcf80 ) , but other cf cards should work too ) . a laptop or pc with flash card reader and the data analysis management software ( dams ) suite installed . music player with children 's stories and headphones and a small self - inflatable air mattress are optional . check the time and date settings on the laptop / pc , since these will be recorded as metadata on your files . put the empty memory card and full batteries in the vu - ams device ( successful placement is signalled by a triple beep ) . when the device is on standby , the green light will flash twice every ten seconds . now connect the device to the laptop using the provided cable and start up the dams program . initiate communication with the device ( select the tab ' device ' and choose the appropriate connection mode , infrared cable or bluetooth ) . where the electrodes will be placed , clean the skin with alcohol - wipes , and place the seven electrodes on the chest and back ( figure 1 ) . then attach the lead wires following the color scheme , and connect them to the device . check the battery type and battery voltage indication ( this should be about 3.4 v for alkaline and about 2.4 v for rechargeable nimh batteries ) . fill out the identification field . measure the distance between the two chest electrodes in millimeters , and fill this out in the field ' icg - v distance ' . then click ' send settings ' to send the current settings / id to the device . now , the ' online ' option of the program should be used to display the ecg , z ( this is the respiration ) and dz / dt ( this is the icg ) . the z signal reflects the base impedance across the thorax , which after appropriate filtering can be used to extract the respiration signal with high fidelity . the dz / dt signal is the z differentiated over time and reflects rapid changes in z linked to the ejection of blood from the ventricle into the aorta . . the r - wave should be upward and it should be the peak with the largest ( absolute ) amplitude in either direction.the z should be within -0.5 and + 0.5 most of the time and dz / dt between -1 and + 1 /sec.z0 should always stay within an 8 to 20 range . this variation reflects the fact that the thorax impedance signal depends on the distance between the measuring electrodes which is a function of the child 's height , and the ' wetness ' of the thorax column enclosed by the measuring electrodes , differences in body composition ( e.g. bmi ) can affect the amplitude of the dz / dt signal ( fat mass containing less water than muscle ) . individual differences in absolute z0 amplitude are also reflected in the z signal but this does not affect the determination of systolic time intervals , which are amplitude-independent.the z signal should reflect deep breathing of the subject clearly ( instruct the child to take a slow deep breath and exhale slowly).in the icg the typical upward waveform reflecting the cardiac ejection phase should be clearly detectable . if these criteria are not met , re - clean the skin and re - attach the electrodes until satisfactory signals are obtained . a clear qrst - complex should be detectable in the ecg . the r - wave should be upward and it should be the peak with the largest ( absolute ) amplitude in either direction . the z should be within -0.5 and + 0.5 most of the time and dz / dt between -1 and + 1 /sec . this variation reflects the fact that the thorax impedance signal depends on the distance between the measuring electrodes which is a function of the child 's height , and the ' wetness ' of the thorax column enclosed by the measuring electrodes , differences in body composition ( e.g. bmi ) can affect the amplitude of the dz / dt signal ( fat mass containing less water than muscle ) . individual differences in absolute z0 amplitude are also reflected in the z signal but this does not affect the determination of systolic time intervals , which are amplitude - independent . the z signal should reflect deep breathing of the subject clearly ( instruct the child to take a slow deep breath and exhale slowly ) . in the icg the typical upward waveform reflecting the cardiac ejection phase should be clearly detectable . if these criteria are not met , re - clean the skin and re - attach the electrodes until satisfactory signals are obtained . when good signals are attained , start data recording by pressing the ' start ' button . you will hear a beep acknowledging the start of the recording and the green light will start flashing once every three seconds . the registration has now started . once the registration has started , ask the child to lie down for the first experimental condition . when the child has been in the supine position ( without head - up tilt ) for two minutes , you shortly ( < 2 s ) press the small black button on top of the device . pressing this button marks a special event , and will later on help you identify the start of this condition in your data . now have the child sit up and repeat the procedure for this second condition . press the children are instructed to rest quietly during these conditions . to stop the measurement , press and the light will flash every 10 sec to indicate it has stopped and is in ' stand by ' mode . once the device has stopped , you may disconnect the lead wire plug from the connector and the lead wires from the electrodes . remove the batteries and flash card form the vu - ams device and place the flash card in the reader unit . move the acquired files to a designated directory ( typically the name of the directory will be identical to the subject identifier used in the identification field ) . upon opening the data with vu - dams program , the data will be automatically converted from raw data format ( extension .5fs ) to a new format ( extension .amsdata ) . this is the data file that vu - dams will be using in the ensuing steps . an automated algorithm will detect all r - peaks in the ecg signal and select ( if present ) periods with very low ecg quality for removal . in the upper left hand corner the number of blue ( correct ) , yellow ( medium suspicious ) or red ( highly suspicious ) is indicated . by pressing ' . ' ( dot ) the cursor is moved to the next suspicious r - peak and the user can delete or add markers for r - waves by hand . the main aim is to obtain a mean value for the heart rate , the preejection period ( pep ) and measures of respiratory sinus arrhythmia ( rsa , hf , rmssd ) across the experimental conditions used . therefore proceed by indicating which periods in the raw data correspond to these conditions . this process two panels show the heart rate signal and movement signal respectively , as well as the actual time of the recording . place the mouse cursor in the top bar where it says " click and drag to add labels " at around the start time of your first condition and drag the mouse to the end time of that condition . these times are either obtained from a written record of start and stop times ( that you noted down during data collection ) or you can use the start and stop markers obtained from pressing the button at the start and end of each condition , which are the vertical lines running across the hr and movement graphs . each label vu - dams needs to be made aware of the experimental design by a so - called label configuration file ( label.cfg ) . this is an ascii file that can be opened with most text editors and , for example , looks like this : # exp_condition 10 lying down 11 sitting up by placing the label.cfg file in the directory of the .amsdata files , it will be automatically loaded by the vu - dams program . once a label has been made , a pop up screen will appear with the categories / values listed in the label.cfg file . select ' lying down ' for the first label and ' sitting up ' for the second label . after labeling , select the ' impedance scoring ' tab to score the pep in the impedance cardiogram . for each of the conditions an ensemble averaged dz / dt waveform place the four vertical cursors in the correct positions : ecg q - wave onset ( start of electrical activity ) , icg b - point ( start of the ejection phase ) , icg dz / dt - min ( maximal ejection speed ) , and icg x - point ( aortic valve closure - end of ejection phase ) . next , select the ' respiration scoring ' tab to score the peak - valley rsa using the respiratory and ecg signals . automated breath - to - breath scoring of the respiratory interval and the shortest interbeat interval during inspiration and the longest interbeat interval during expiration can now be inspected . typically the automated detection algorithm should not classify more than 15% of the breaths as deviant - otherwise inspect the respiration signal and tune the parameters of the detection algorithm as needed . guidelines for visual inspection of the ecg , icg and respiration signals and interactive pep and rsa scoring can be found on the vu - ams website , www.vu-ams.nl . each row represents the average value of a series of physiological parameters ( heart rate , pep , rsa , rr ) for each labeled time period . the last column indicates the values of all categories used during labeling ( here only a single category ' experimental condition ' with two values , ' sitting up ' and ' lying down ' ) . spectral powers of the interbeat interval times series are given only for labels with a minimum length of 4 min ( otherwise the missing code is displayed ) . the spreadsheet in this display can be exported to ascii or excel for further statistical analyses . you need : a vu - ams5fs ambulatory recording device ( including an infrared interface cable that either connects to the rs232 serial port of a pc or to a usb port).7 electrodes ( we used conmed 1690 - 003).2 charged aa-batteries.an empty compactflash memory card ( the vu - ams5fs has been extensively tested with the 1 gb 80x cf card from transcend ( ts1gcf80 ) , but other cf cards should work too).a laptop or pc with flash card reader and the data analysis management software ( dams ) suite installed.a stopwatch.music player with children 's stories and headphones and a small self - inflatable air mattress are optional . a vu - ams5fs ambulatory recording device ( including an infrared interface cable that either connects to the rs232 serial port of a pc or to a usb port ) . 7 electrodes ( we used conmed 1690 - 003 ) . 2 charged aa - batteries . an empty compactflash memory card ( the vu - ams5fs has been extensively tested with the 1 gb 80x cf card from transcend ( ts1gcf80 ) , but other cf cards should work too ) . a laptop or pc with flash card reader and the data analysis management software ( dams ) suite installed . music player with children 's stories and headphones and a small self - inflatable air mattress are optional . check the time and date settings on the laptop / pc , since these will be recorded as metadata on your files . put the empty memory card and full batteries in the vu - ams device ( successful placement is signalled by a triple beep ) . when the device is on standby , the green light will flash twice every ten seconds . now connect the device to the laptop using the provided cable and start up the dams program . initiate communication with the device ( select the tab ' device ' and choose the appropriate connection mode , infrared cable or bluetooth ) . where the electrodes will be placed , clean the skin with alcohol - wipes , and place the seven electrodes on the chest and back ( figure 1 ) . then attach the lead wires following the color scheme , and connect them to the device . check the battery type and battery voltage indication ( this should be about 3.4 v for alkaline and about 2.4 v for rechargeable nimh batteries ) . fill out the identification field . measure the distance between the two chest electrodes in millimeters , and fill this out in the field ' icg - v distance ' . then click ' send settings ' to send the current settings / id to the device . now , the ' online ' option of the program should be used to display the ecg , z ( this is the respiration ) and dz / dt ( this is the icg ) . the z signal reflects the base impedance across the thorax , which after appropriate filtering can be used to extract the respiration signal with high fidelity . the dz / dt signal is the z differentiated over time and reflects rapid changes in z linked to the ejection of blood from the ventricle into the aorta . . the r - wave should be upward and it should be the peak with the largest ( absolute ) amplitude in either direction.the z should be within -0.5 and + 0.5 most of the time and dz / dt between -1 and + 1 /sec.z0 should always stay within an 8 to 20 range . this variation reflects the fact that the thorax impedance signal depends on the distance between the measuring electrodes which is a function of the child 's height , and the ' wetness ' of the thorax column enclosed by the measuring electrodes , differences in body composition ( e.g. bmi ) can affect the amplitude of the dz / dt signal ( fat mass containing less water than muscle ) . individual differences in absolute z0 amplitude are also reflected in the z signal but this does not affect the determination of systolic time intervals , which are amplitude-independent.the z signal should reflect deep breathing of the subject clearly ( instruct the child to take a slow deep breath and exhale slowly).in the icg the typical upward waveform reflecting the cardiac ejection phase should be clearly detectable . if these criteria are not met , re - clean the skin and re - attach the electrodes until satisfactory signals are obtained . a clear qrst - complex should be detectable in the ecg . the r - wave should be upward and it should be the peak with the largest ( absolute ) amplitude in either direction . the z should be within -0.5 and + 0.5 most of the time and dz / dt between -1 and + 1 /sec . this variation reflects the fact that the thorax impedance signal depends on the distance between the measuring electrodes which is a function of the child 's height , and the ' wetness ' of the thorax column enclosed by the measuring electrodes , differences in body composition ( e.g. bmi ) can affect the amplitude of the dz / dt signal ( fat mass containing less water than muscle ) . individual differences in absolute z0 amplitude are also reflected in the z signal but this does not affect the determination of systolic time intervals , which are amplitude - independent . the z signal should reflect deep breathing of the subject clearly ( instruct the child to take a slow deep breath and exhale slowly ) . in the icg the typical upward waveform reflecting the cardiac ejection phase should be clearly detectable . if these criteria are not met , re - clean the skin and re - attach the electrodes until satisfactory signals are obtained . when good signals are attained , start data recording by pressing the ' start ' button . you will hear a beep acknowledging the start of the recording and the green light will start flashing once every three seconds . the registration has now started . once the registration has started , ask the child to lie down for the first experimental condition . when the child has been in the supine position ( without head - up tilt ) for two minutes , you shortly ( < 2 s ) press the small black button on top of the device . pressing this button marks a special event , and will later on help you identify the start of this condition in your data . after four minutes , press the event button again . now have the child sit up and repeat the procedure for this second condition . press the children are instructed to rest quietly during these conditions . to stop the measurement , press and the light will flash every 10 sec to indicate it has stopped and is in ' stand by ' mode . once the device has stopped , you may disconnect the lead wire plug from the connector and the lead wires from the electrodes . remove the batteries and flash card form the vu - ams device and place the flash card in the reader unit . move the acquired files to a designated directory ( typically the name of the directory will be identical to the subject identifier used in the identification field ) . upon opening the data with vu - dams program , the data will be automatically converted from raw data format ( extension .5fs ) to a new format ( extension .amsdata ) . this is the data file that vu - dams will be using in the ensuing steps . an automated algorithm will detect all r - peaks in the ecg signal and select ( if present ) periods with very low ecg quality for removal . in the upper left hand corner the number of blue ( correct ) , yellow ( medium suspicious ) or red ( highly suspicious ) is indicated . by pressing ' . ' ( dot ) the cursor is moved to the next suspicious r - peak and the user can delete or add markers for r - waves by hand . the main aim is to obtain a mean value for the heart rate , the preejection period ( pep ) and measures of respiratory sinus arrhythmia ( rsa , hf , rmssd ) across the experimental conditions used . therefore proceed by indicating which periods in the raw data correspond to these conditions . this process two panels show the heart rate signal and movement signal respectively , as well as the actual time of the recording . place the mouse cursor in the top bar where it says " click and drag to add labels " at around the start time of your first condition and drag the mouse to the end time of that condition . these times are either obtained from a written record of start and stop times ( that you noted down during data collection ) or you can use the start and stop markers obtained from pressing the button at the start and end of each condition , which are the vertical lines running across the hr and movement graphs . each label can be given a ( unique ) identifier to signal a particular condition . in our case vu - dams needs to be made aware of the experimental design by a so - called label configuration file ( label.cfg ) . this is an ascii file that can be opened with most text editors and , for example , looks like this : # exp_condition 10 lying down 11 sitting up by placing the label.cfg file in the directory of the .amsdata files , it will be automatically loaded by the vu - dams program . once a label has been made , a pop up screen will appear with the categories / values listed in the label.cfg file . select ' lying down ' for the first label and ' sitting up ' for the second label . after labeling , select the ' impedance scoring ' tab to score the pep in the impedance cardiogram . for each of the conditions an ensemble averaged dz / dt waveform place the four vertical cursors in the correct positions : ecg q - wave onset ( start of electrical activity ) , icg b - point ( start of the ejection phase ) , icg dz / dt - min ( maximal ejection speed ) , and icg x - point ( aortic valve closure - end of ejection phase ) . next , select the ' respiration scoring ' tab to score the peak - valley rsa using the respiratory and ecg signals . automated breath - to - breath scoring of the respiratory interval and the shortest interbeat interval during inspiration and the longest interbeat interval during expiration can now be inspected . typically the automated detection algorithm should not classify more than 15% of the breaths as deviant - otherwise inspect the respiration signal and tune the parameters of the detection algorithm as needed . guidelines for visual inspection of the ecg , icg and respiration signals and interactive pep and rsa scoring can be found on the vu - ams website , www.vu-ams.nl . each row represents the average value of a series of physiological parameters ( heart rate , pep , rsa , rr ) for each labeled time period . the last column indicates the values of all categories used during labeling ( here only a single category ' experimental condition ' with two values , ' sitting up ' and ' lying down ' ) . spectral powers of the interbeat interval times series are given only for labels with a minimum length of 4 min ( otherwise the missing code is displayed ) . the spreadsheet in this display can be exported to ascii or excel for further statistical analyses . in the amsterdam born children and their development study , a dutch prospective , longitudinal birth cohort , the measurement protocol was started in 3,097 children . approval was obtained from the academic medical center medical ethical committee , the vu university medical center medical ethical committee and the registration committee of amsterdam . all participating mothers gave written informed consent for themselves and their children . as the monitors are lightweight and unobtrusive , the children tolerated these measurements very well . we do not have data on the refusal rate , but experience taught us that only a few children resisted the placement of the electrodes and thereby obstructed further assessment . of the 3,097 registrations , 0.7% were lost due to either equipment failure or misplacement of files . out of the 3,074 registrations left , 98.7% were of children who completed the entire protocol ( n = 3,056 ) . within each of the labelled time periods ( we originally labelled four time periods , but later summarized these to two ) , we encountered unclear icg signals , meaning pep could not be determined . this led to a loss of 1.5% in the first out of four labelled periods , 2.4% in the second , 2.8% in the third and 4.1% in the fourth period . complete data on pep in all time periods was available in 2,797 cases ( 91.5% , thus 8.5% loss due to unclear icg signals ) . complete data on heart rate ( hr ) , pre - ejection period ( pep ) and respiratory sinus arrhythmia ( rsa ) , as well as sex and age , was available from 2,761 children ; in this final step , 1.3% data loss occurred , due to unknown reasons . the mean age of the children was 5.7 years ( sd 0.5 ; interquartile range 5.0:6.5 ) , and their bmi was 15.5 kg / m ( sd 1.5 ; interquartile range 13.9:17.2 ) . the mean values of the major outcome variables hr , pep , and rsa are given in table 1 and graphically depicted in figure 3 , separately for boys and girls . hr ( both lying down and sitting up ) and pep ( only sitting up ) were higher in girls than in boys ( both postures ) . the higher values for hr in girls are likely to be caused by the lower vagal ( parasympathetic ) cardiac control . their sympathetic cardiac control was not different or even lower than that in boys ( sitting up ) . in both sexes , hr was higher when sitting up compared to lying down , whereas rsa was lower when sitting up . this effect was also as expected , and it reflects the outcome of opposite processes : lower sympathetic activity ( lengthens pep ) while lying down with increased preload ( shortens pep ) . table 1 . cardiac autonomic nervous system measures in boys and girls , by posture on posture difference . p < 0.05 for one sample t - test on sex difference . p < 0.05 for paired samples t - test . the seven electrodes should be placed on the participant 's chest and back . the first ecg electrode ( v- ) is placed slightly below the right collar bone 4 cm to the right of the sternum . the second ecg electrode ( v+ ) is placed at the apex of the heart over the ninth rib on the left lateral margin of the chest approximately at the level of the processus xiphodius . the third ecg electrode ( gnd ) is a ground electrode and is placed on the right side , between the lower two ribs at the right abdomen . the first icg measuring electrode ( v1 ) is placed at the top end of the sternum , between the tips of the collar bones . the second icg measuring electrode is placed at the xiphoid complex of the sternum , where the ribs meet . the two current electrodes are placed on the back : i- on the spine over the cervical vertebra c4 , at least 3 cm ( 1 in ) above the icg measuring elec - trode v- , and i+ between thoracic vertebrae t8 and t9 on the spine , at least 3 cm ( 1 " ) below the icg measuring elec - trode v2 . the icg electrode placement takes into account that the largest part of the left ventricle driven change in thorax impedance is captured by the column between the suprasternal notch and the processus xiphoideus . the typical settings used for a recording as displayed by the dams software after connecting to the vu - ams5fs device . figure 3 . cardiac autonomic nervous system measures in boys and girls , by posture . we used an ambulatory recording device to measure cardiac autonomic control in 3097 children , aged between 5 and 7 years . seven electrodes sufficed to measure the ecg and icg from which the heart rate , heart rate variability and the systolic time intervals were extracted . heart rate variability in the respiratory frequency band ( rsa ) is a valid indicator of cardiac parasympathetic activity . the systolic time interval , pep , by reflecting cardiac contractility , is a valid indicator of cardiac sympathetic activity . the mean values obtained for hr , pep and rsa , the effects of posture changes and the differences between boys and girls were in line with what would be expected from the literature . as ambulatory monitoring removed the necessity of assessment in a laboratory our recordings could be done in various locations ( e.g. school , sports center , science museum ) without differences in signal recording quality . however , it is of crucial importance to standardize within or between subject comparisons for posture and physical load , as afterload and preload effects can co - determine the pep without any changes in cardiac sympathetic drive . we conclude that ambulatory recording of the ecg and icg in large samples of children is highly feasible and propose the current standardized study design as a useful template for future assessments of cardiac autonomic control in children .

the autonomic nervous system ( ans ) controls mainly automatic bodily functions that are engaged in homeostasis , like heart rate , digestion , respiratory rate , salivation , perspiration and renal function . the ans has two main branches : the sympathetic nervous system , preparing the human body for action in times of danger and stress , and the parasympathetic nervous system , which regulates the resting state of the body.ans activity can be measured invasively , for instance by radiotracer techniques or microelectrode recording from superficial nerves , or it can be measured non - invasively by using changes in an organ 's response as a proxy for changes in ans activity , for instance of the sweat glands or the heart . invasive measurements have the highest validity but are very poorly feasible in large scale samples where non - invasive measures are the preferred approach . autonomic effects on the heart can be reliably quantified by the recording of the electrocardiogram ( ecg ) in combination with the impedance cardiogram ( icg ) , which reflects the changes in thorax impedance in response to respiration and the ejection of blood from the ventricle into the aorta . from the respiration and ecg signals , respiratory sinus arrhythmia can be extracted as a measure of cardiac parasympathetic control . from the ecg and the left ventricular ejection signals , the preejection period can be extracted as a measure of cardiac sympathetic control . ecg and icg recording is mostly done in laboratory settings . however , having the subjects report to a laboratory greatly reduces ecological validity , is not always doable in large scale epidemiological studies , and can be intimidating for young children . an ambulatory device for ecg and icg simultaneously resolves these three problems.here , we present a study design for a minimally invasive and rapid assessment of cardiac autonomic control in children , using a validated ambulatory device 1 - 5 , the vu university ambulatory monitoring system ( vu - ams , amsterdam , the netherlands , www.vu-ams.nl ) .

Protocol 1. Preparation: Starting Up 2. The Registration Period 3. Processing the Data Representative Results Discussion Disclosures

PMC3597427

our visits were part of our lab time , which unfortunately meant that we were interacting with the second graders at the end of their day . activities . all activities chosen except for the brain puzzle were adapted from brain - related activities found on dr . . we chose activities using the following criteria : they needed to be time - flexible , fun , and cheap . activities needed to be able to be included in a modular fashion , depending on how much time we had in the classroom and on the abilities of the students on that particular day . since we knew that we would be reaching almost 500 students , any disposables our biggest expense was the brain puzzle , because we wanted to make enough so that each student could have one and take it home . we constructed 500 brain puzzles using recycled manila folders , brain images , simple text about basic functions of the brain , plastic ziplock bags , tape and glue . we found a colorful labeled brain image on the web ( many exist ; we used one from http://www.sharpbrains.com/blog/2008/06/05/your-brain-on-trading-101/ ) and saved costs by printing them out four to a page . we cut each brain into 710 pieces and placed the pieces into a ziplock bag which was then taped into the inside of the folder . we typically did the brain puzzle as the first activity , after introductions and the discussion about college . once the second graders had assembled the puzzle , the denison students briefly described and answered questions about the parts of the brain ( fig . 1 ) . depending on the time available and the focus of the second graders , this activity took between 10 and 20 minutes . afterwards , when the second graders did different activities relating to their senses or to movement , we related these experiences to the relevant parts of the brain . the goal of the mystery sock activity was to have students identify common objects using their sense of touch , which we then related back to the parietal lobe . our mystery socks consisted of plain white sports socks filled with items that would present some interesting tactile contrasts and that were objects that i had around the house : markers , glue sticks , whiffle balls , playdough , pennies , legos , small metal trucks and cars , and tissues . each denison team had the same assortment of eight socks . during the activity , each child tried to guess the contents of each sock by using their sense of touch alone . the hardest part of this activity was keeping the second graders from shouting out what they thought was in the sock . but by asking them to keep it a secret , we were able to keep the experience novel for everyone . students really enjoyed guessing once everyone had felt all of the socks . a good strategy for keeping track of which students had felt each sock was to have the students form lines in front of the denison students , who were holding the socks ( fig . the goal of the jelly bean activity was for students to recognize the extent to which taste is affected by smell , and thus also the general concept of how the senses can work together . for our jellybean experiments , we used lifesaver jellybeans because they are inexpensive and have strong flavors and fragrances that most people enjoyed . typically , denison students ran this exercise by sorting the jellybeans by color and giving all of the second graders the same color jellybean . during the first round , the second graders were allowed to use all their senses to identify the flavor . during the second round they were asked to close their eyes , to avoid the color cue , and for the third round , they were asked to pinch their nose to minimize the flavor - amplifying effect of odor . the mystery noises activity involved producing noises which the second graders had to identify . in some cases , denison students were able to play electronic noises of ice cream trucks , cows mooing , popcorn popping , etc . on their laptops . these sounds were all found on www.youtube.com . when the internet was not available in the classrooms , denison students created sounds by ripping paper , clapping erasers , dropping a penny , closing a door , etc . the goal of this exercise was to show how signals can be passed from one neuron to another . students formed two or three lines with equal numbers of students and were instructed to tap the next person in line once they felt a tap themselves . a tagged player must hold the hand of the first player and together they have to chase the other players . as more and more players are tagged , they are added to the chain of neurons . the game ends when all of the players are part of the chain . student engagement . over the course of each activity , the denison student assigned to monitor second grader engagement observed each student in the classroom . this was facilitated by the fact that for activities 15 students were typically grouped at desk clusters or in rows . they noted each student s gender and decided if he / she was engaged or unengaged and recorded this in a data sheet with a + or a - mark . students were considered engaged when they watched the denison students leading the activity , followed directions , interacted with peers when instructed ( as in the tag and message games ) , and worked independently as assigned ( as in the brain puzzle ) , etc . students were considered unengaged when they were not looking at the students leading the activities or were talking out of turn , had their head down on their desk , or were otherwise not following directions . students who received mostly for an activity were given a 1 for that activity , students who received an approximately equal mix of and + were given a 2 , and students who received mostly + were considered to have an engagement of 3 for that activity . since these scores are ordinal , we compared student engagement between boys and girls using the mann - whitney test with the improved normal approximation ( zar , 1999 ) . this test was calculated busing jmp 8 software ( sas institute inc . , 2008 ) . effect sizes were calculated using cohen s d statistics , in which the difference between the means is divided by the mean standard deviation ( cohen , 1988 ) . denison students also filled out a weekly individual form reporting on how the activities went and what they learned . they also filled out an anonymous survey commenting on the service learning experience as a whole . our visits were part of our lab time , which unfortunately meant that we were interacting with the second graders at the end of their day . activities . all activities chosen except for the brain puzzle were adapted from brain - related activities found on dr . . we chose activities using the following criteria : they needed to be time - flexible , fun , and cheap . activities needed to be able to be included in a modular fashion , depending on how much time we had in the classroom and on the abilities of the students on that particular day . since we knew that we would be reaching almost 500 students , any disposables our biggest expense was the brain puzzle , because we wanted to make enough so that each student could have one and take it home . we constructed 500 brain puzzles using recycled manila folders , brain images , simple text about basic functions of the brain , plastic ziplock bags , tape and glue . we found a colorful labeled brain image on the web ( many exist ; we used one from http://www.sharpbrains.com/blog/2008/06/05/your-brain-on-trading-101/ ) and saved costs by printing them out four to a page . we cut each brain into 710 pieces and placed the pieces into a ziplock bag which was then taped into the inside of the folder . we typically did the brain puzzle as the first activity , after introductions and the discussion about college . once the second graders had assembled the puzzle , the denison students briefly described and answered questions about the parts of the brain ( fig . 1 ) . depending on the time available and the focus of the second graders , this activity took between 10 and 20 minutes . afterwards , when the second graders did different activities relating to their senses or to movement , we related these experiences to the relevant parts of the brain . the goal of the mystery sock activity was to have students identify common objects using their sense of touch , which we then related back to the parietal lobe . our mystery socks consisted of plain white sports socks filled with items that would present some interesting tactile contrasts and that were objects that i had around the house : markers , glue sticks , whiffle balls , playdough , pennies , legos , small metal trucks and cars , and tissues . each denison team had the same assortment of eight socks . during the activity , each child tried to guess the contents of each sock by using their sense of touch alone . the hardest part of this activity was keeping the second graders from shouting out what they thought was in the sock . but by asking them to keep it a secret , we were able to keep the experience novel for everyone . students really enjoyed guessing once everyone had felt all of the socks . a good strategy for keeping track of which students had felt each sock was to have the students form lines in front of the denison students , who were holding the socks ( fig . the goal of the jelly bean activity was for students to recognize the extent to which taste is affected by smell , and thus also the general concept of how the senses can work together . for our jellybean experiments , we used lifesaver jellybeans because they are inexpensive and have strong flavors and fragrances that most people enjoyed . typically , denison students ran this exercise by sorting the jellybeans by color and giving all of the second graders the same color jellybean . during the first round , the second graders were allowed to use all their senses to identify the flavor . during the second round they were asked to close their eyes , to avoid the color cue , and for the third round , they were asked to pinch their nose to minimize the flavor - amplifying effect of odor . the mystery noises activity involved producing noises which the second graders had to identify . in some cases , denison students were able to play electronic noises of ice cream trucks , cows mooing , popcorn popping , etc . on their laptops . these sounds were all found on www.youtube.com . when the internet was not available in the classrooms , denison students created sounds by ripping paper , clapping erasers , dropping a penny , closing a door , etc . the goal of this exercise was to show how signals can be passed from one neuron to another . students formed two or three lines with equal numbers of students and were instructed to tap the next person in line once they felt a tap themselves . a tagged player must hold the hand of the first player and together they have to chase the other players . as more and more players are tagged , they are added to the chain of neurons . the game ends when all of the players are part of the chain . student engagement . over the course of each activity , the denison student assigned to monitor second grader engagement observed each student in the classroom . this was facilitated by the fact that for activities 15 students were typically grouped at desk clusters or in rows . they noted each student s gender and decided if he / she was engaged or unengaged and recorded this in a data sheet with a + or a - mark . students were considered engaged when they watched the denison students leading the activity , followed directions , interacted with peers when instructed ( as in the tag and message games ) , and worked independently as assigned ( as in the brain puzzle ) , etc . students were considered unengaged when they were not looking at the students leading the activities or were talking out of turn , had their head down on their desk , or were otherwise not following directions . students who received mostly for an activity were given a 1 for that activity , students who received an approximately equal mix of and + were given a 2 , and students who received mostly + were considered to have an engagement of 3 for that activity . since these scores are ordinal , we compared student engagement between boys and girls using the mann - whitney test with the improved normal approximation ( zar , 1999 ) . this test was calculated busing jmp 8 software ( sas institute inc . , 2008 ) . effect sizes were calculated using cohen s d statistics , in which the difference between the means is divided by the mean standard deviation ( cohen , 1988 ) . denison students also filled out a weekly individual form reporting on how the activities went and what they learned . they also filled out an anonymous survey commenting on the service learning experience as a whole . we successfully visited all 24 second grades in newark , oh , and interacted with about 560 second graders . the activities presented in each classroom varied depending on teacher preparedness , time available and other constraints ( such as achievement testing occurring elsewhere in the building ) . every visit included the brain puzzle and the jelly bean taste test , and most groups did the mystery socks , but fewer groups did the other activities . all activities except for message transmission had a score of at least 2.5 out of 3 , with the mean engagement for all activities being 2.63 . the most engaging activity , for both boys and girls , was the neuron chain tag ( 2.77 and 2.81 for boys and girls ) . a summary of the mean engagement scores of the six activities , analyzed by gender , is shown in figure 3 . when student engagement was analyzed as a function of gender , effect sizes measured as cohen s d statistics for these activities were small to moderate ( 0.203 and 0.285 ) . the other four activities ( mystery socks , mystery noises , message , and neuron chain tag ) showed no difference in engagement between male and female second graders , although the message activity might well show a difference with a larger sample size . the first hypothesis , that girls would be more engaged in the brain puzzle than boys was supported ( p < 0.0226 , d = 0.203 ; figure 3 , table 1 ) . the second hypothesis , that the sensory activities would be gender neutral , was supported by two out of the three sensory activities ( mystery socks and mystery noises ) , but not by jelly beans ( p < 0.0074 , d = 0.285 ) . the third hypothesis , that the more active games would engage boys more than girls , was not supported . message transmission and neuron chain tag - the favorite of both boys and girls - was gender neutral . it is worth considering the biases that could have crept in to the observations of the effect of gender on student engagement . although our class did not explicitly discuss the theoretical gender bias of our chosen activities , saying only that we wanted to have a variety of types of activities to accommodate different learning styles , these students may have formed predictions and this may have affected their scoring . i tried to minimize the likelihood of bias by creating a rubric for scoring student engagement that relied on simple , clear observations . i was initially surprised that girls were equally or more engaged than boys for all six activities . however , this could be due to the fact that engagement has to do with following instructions and looking at whoever is giving instructions . perhaps , if engagement had been structured to be about enthusiasm , jumping out of one s seat , asking questions , and interrupting out of eagerness , boys might have received higher scores . this experiment could be the subject of an outreach in another offering of this course . eighteen out of 23 students ( 78% ) thought that the experience of teaching about college and about the brain enhanced their understanding of the content material , saying that they learned more about the brain and [ its ] functions by teaching 2nd graders . typical responses from the 22% who did not feel that this experience helped them directly with course content included that the experience did nt enhance class subject matter but [ they ] did learn more about kids and learning styles . twenty - one out of 23 students ( 91% ) felt that the experience gave them a window into solving community problems , saying that this outreach exemplifies and supports denison s mission and that they felt proud to have taken action . the two who did not respond affirmatively felt that the college awareness part was certainly important , but did nt think that greater brain awareness could solve this community s problems . twenty out of 23 students ( 87% ) felt that they had developed a greater sense of themselves as agents of change as a result of the experience . the three who did not respond affirmatively felt that earlier volunteering experiences had transformed them already . i feel like i may have left a lasting impression on students who may otherwise get little attention , and i became passionate again about volunteering . some students reflected that the experience made me realize that people in the surrounding community do nt have the great opportunities and constant encouragement that i do . they noted that simple things , like sitting at the table with the second graders , and giving them a lot of attention and encouragement , made a big difference . some comments included : this definitely enhanced my denison experience , the peak program [ was ] extremely beneficial to both volunteers and participants , they wanted to stay longer to have more impact , and that the outreach made me examine myself as a teacher and as a student at the same time . i think that my students , as well as the second graders , were highly engaged . i think that the denison students benefitted from seeing male - female differences in learning styles and from analyzing and discussing the data . i think that they also benefitted personally - either finding a drive to volunteer , or perhaps realizing how fortunate they were . altogether , we devoted six out of our fourteen lab times to this outreach ( one planning , one preparing and practice , four weeks in the classrooms ) . part of this extensive time commitment was due to the mission of peak and the program s desire to have every student in the targeted grade level uniformly involved in the outreach . since this class was a non - majors class , i felt that the benefits of this experience outweighed the costs ( losing time for additional wetlabs or independent projects ) . i am not sure that i could rationalize using the same amount of time doing outreach for my upper level neurophysiology course . after this experience , however , i will be looking for ways that we can reach out to the community in a different way for a shorter period of time . supplementary material : script with lesson plans for each activity ( 1 ) , student engagement forms ( 2 ) , and lab supply list ( 3 ) .

this article describes a neuroscience outreach program developed by college undergraduates and aimed at second graders . over a period of four weeks , twenty - five denison students enrolled in a non - majors course on gender and the brain visited twenty - four second grade classrooms to engage a total of 464 students . we had a mission to both promote college awareness and to specifically bring some brain science into the classroom . the desire to engage students with the brain was in part a wish to celebrate brain awareness week and in part a wish to follow a feminist tenet of bridging theory and practice via activism . the college students chose six activities : a brain puzzle , a sock content guessing game , a jelly bean olfaction and taste test , mystery noises , a message transmission game , and a version of tag . during our outreach with the second graders , my students monitored student engagement and compared engagement between male and female second graders . engagement was high for nearly all activities but girls were more engaged than boys during the brain puzzle and jelly bean activities . effect sizes measured as cohen s d statistics were small to large ( 0.2 to 0.93 ) . the other four activities ( mystery socks , mystery noises , message transmission and neuron chain tag ) showed no difference in engagement between male and female second graders . our program benefited the denison students as well , introducing many to community involvement and awakening in them an interest in teaching or working with kids .

MATERIALS AND METHODS Basic plan RESULTS AND DISCUSSION None

PMC3860139

the cochlear implant ( ci ) transforms acoustic signals from the environment into electric impulses , which are then used to stimulate intact fibers of the auditory nerve . with this treatment , individuals with profound hearing loss ( hl ) current technology and speech processing strategies allow many ci recipients to achieve impressive accuracy in open - set speech recognition , and the ci is arguably the most effective neural prosthesis ever developed [ 13 ] . however , the success of the outcome depends both on duration of deafness prior to implantation [ 4 , 5 ] and on the onset of deafness before ( prelingually ) [ 47 ] or after ( postlingually ) critical stages in the acquisition of language . in many cases , the greatest gains of performance occur in the first three months of use [ 911 ] . the dramatic improvements following implantation not only demonstrate the efficiency of the ci technology , but also point to the role of cortical plasticity as a means to reactivate brain function . plasticity is a term used to describe the reorganization of the central nervous system by means of synaptic changes and rewiring of neural circuits . in cases of cochlear implantation , neural plasticity associated with deprivation of auditory input and adaptation to the absence of stimuli is of particular interest . reduced input to the brain from impaired auditory pathways results in significant changes in the central auditory system and is accompanied by a recruitment of deprived cortices in response to input from the intact senses [ 1317 ] . when auditory input to the brain is reintroduced , this novel auditory experience may itself induce additional plasticity . the sensory reafferentation provided by the ci thus offers a unique opportunity to study the effects of preceding deafness on functional brain organization . in normal - hearing ( nh ) adults , language processing is associated with extensive frontal activation in the left cerebral hemisphere , including the anterior ( brodmann 's areas ( ba ) 45 and 47 ) and posterior ( ba 44 and 45 ) parts of the left inferior frontal gyrus ( lifg ) , the latter often referred to as broca 's area [ 19 , 20 ] . traditionally , this area is mainly assigned an expressive language function , but several studies show a relationship between the perception of language and left frontal activity , both when stimuli are presented aurally [ 2124 ] and visually [ 2428 ] . neuroimaging experiments comparing auditory responses of ci users and normal - hearing control participants , while listening to speech or complex nonspeech , generally reveal bilateral activity in the primary and secondary auditory cortices , including both superior and middle temporal gyri [ 12 , 2934 ] . one consistent outcome of these studies is the more dominant right temporal activity of ci users listening to speech , that is , the observation of more bilateral activity than would be expected on the basis of the classical presumption of left - lateralized activity of language processing in normal - hearing . however , in these studies , activation of other classic language regions such as broca 's area was not a consistent finding . naito and colleagues found broca 's area to be activated only when the ci participants silently repeated sentences [ 31 , 36 ] . compared brain activity in experienced ci users according to their levels of speech comprehension performance . they found that , unlike ci users with low speech comprehension , single words and speech yielded raised activity in the left inferior prefrontal cortex ( lipc ) in ci users with excellent speech perception . some observed activations outside the classic language areas , including anterior cingulate , parietal regions , and left hippocampus , have been attributed to nonspecific attentional mechanisms and memory in ci users [ 31 , 32 ] . furthermore , some studies have reported convincing evidence of visual activity in response to auditory stimuli or auditory activity in response to visual stimuli in ci users . although much debate about the identity of the brain systems that are changed and the mechanisms that mediate these changes exists , the general belief is that this cross - modal reorganization is associated with the strong visual speech - reading skills developed by ci users during the period of deafness , which are maintained or even improved after cochlear implantation , despite progressive recovery of auditory function [ 5 , 11 , 33 , 3841 ] . the possible reasons for these mixed results may include differences in experimental paradigms , small sample sizes , heterogeneous populations , variance in statistical thresholds , and a lack of longitudinal control of plasticity . with the present study , we tested the cortical mechanisms underlying the restoration of hearing and speech perception in the first six - month period following implant switch - on . we expected to see inactive neuronal pathways reactivated in ci recipients , within three to six months of switch - on , and engagement of cortical areas resembling those of normal - hearing control participants . furthermore , in previous findings , notwithstanding , we expected to see broca 's area involved in speech perception . finally , we expected to see a difference in the progress of adaptation and the involvement of cortical areas between ci users with postlingual hearing loss and ci users with prelingual hearing loss . over the course of two years , patients who were approved for implantation were contacted by mail and invited to take part in the research project , including positron emission tomography ( pet ) and speech perception measures . from a total of 41 patients , 15 accepted and were included in the study ( 6 women , 9 men , mage = 51.8 , age , range : 2173 years ) . four participants had a prelingual onset of hl , indicated by their estimated age at onset of deafness and main use of signed language as communicative strategy . the remaining 11 participants had a postlingual or progressive onset of hl , as indicated by their main use of residual hearing , supported by lip - reading . in accordance with local practice , all ci participants followed standard aural / oral therapy for six months in parallel with the study . the therapy program includes weekly one - hour individually adapted sessions and trains speech perception and articulation . table 1 lists the demographic and clinical data for the 15 participants . to obtain a normal - hearing reference , we recruited a group of nh adults ( 4 women , 2 men , mage = 54.29 years , age range : 4764 years ) for a single pet / test session . all nh participants met the criteria for normal - hearing by passing a full audiometric test . the study was conducted at the pet center , aarhus university hospital , denmark , in accordance with the declaration of helsinki , and approved by the research ethics committee of the central denmark region . nh participants underwent pet once , while ci participants were tested consecutively at three points of time : ( 1 ) within 14 days after switch - on of the implant ( baseline , bl ) , ( 2 ) after three - months ( midpoint , mp ) , and ( 3 ) after six months ( endpoint , ep ) . for purposes of analysis , two subgroups were identified as ( 1 ) the postlingual ( post ) hl subgroup ( n = 11 ) and ( 2 ) the prelingual ( pre ) hl subgroup ( n = 4 ) . the hagerman test is an open - set test which presents sentences organized in lists of ten . the sentences have identical name - verb - number - adjective - noun structures , which the participant is required to repeat . normally , the test is presented in background noise . however , considering the participants ' inexperience with the implant , we removed the background noise . the participants were given one training list with feedback and two trial lists without feedback ( max . sound was played back on a laptop computer through an active loudspeaker ( fostex 6301b , fostex company , japan ) placed in front of the participant . the stimuli were presented at 65 db sound pressure level ( spl ) , and ci users were instructed to use their preferred ci settings during the entire test session and to adjust their processors to a comfortable loudness level . furthermore , participants who used a hearing aid were instructed to turn it off and leave it plugged in . the ci participants performed the hagerman tests in separate sessions at the same milestones as selected for pet data acquisition ( bl , mp , and ep ) . the nh group performed a single hagerman test along with their pet scan session . to identify effects of time , we performed a repeated measures analysis of variance . due to nonnormal distributions in the data , this was done using a nonparametric friedman 's anova in the post and pre subgroups separately . post hoc tests and between - group analyses were performed by mann - whitney nonparametric tests and bonferroni corrected at alpha 0.016 . the hag data were analyzed in spss and plotted with sigmaplot for windows 11.0 ( systat software inc . ) . a high - resolution t1-weighted mr scan was acquired prior to pet scanning . in the case of ci participants , all participants were examined in 2 conditions : ( 1 ) multitalker babble ( bab ) from multiple simultaneous speakers with a complexity close to that of speech and perceived by the listeners as speech - like but devoid of meaning and ( 2 ) running speech ( rs ) , narrating the history of a familiar geographical locality at the rate of 142 words per minute , generated in danish by a standard female voice . the stimuli were played back on a laptop computer in the freeware sound editor software audacity ( http://audacity.sourceforge.net/ ) and delivered directly from the computer 's headphone jack to the external input port of the implant speech processor . bimodally aided participants removed their hearing aid and were fitted with an earplug in the nonimplanted ear during the tomography . these measures were taken to preclude background noise and possible cross - talk from the contralateral ear . the nh participants listened to the stimuli binaurally through a pair of headphones ( akg , k 271 ) . all stimuli were presented at the most comfortable level . to define this level , participants were exposed to the two stimuli once before the tomography . in the tomograph , prior to bolus injection , participants had no information about the nature of the next stimulus , but they were instructed to listen attentively in all cases . after each of the four scans , participants were required to describe what they had heard and , if possible , review the content of the narration . pet . positron emission tomography ( pet ) is a molecular imaging method that yields brain activity , by means of detecting changes in regional cerebral blood flow ( rcbf ) . this is done by computing and comparing the spatial distributions of the uptake of a blood flow tracer . pet measurements are generally limited with respect to spatial and temporal resolution and the invasiveness of the procedure , which requires injection of oxygen-15-labelled water . in this case , however , anatomical and temporal specificity could not have been improved by using functional magnetic resonance imaging ( fmri ) , as the auditory implants are not mri compatible . in addition , pet is an almost noiseless imaging modality , which is useful for both ci participants and for the study of speech . finally , because only the head of the participant is positioned in the tomograph , compared with the whole body imposition of fmri , it is possible to communicate visually with the participant during tomography . we measured raised or reduced cerebral activity as the change of the brain uptake of h2 o oxygen-15-labeled water , which matches the distribution of cerebral blood flow ( cbf ) , using an ecat exact hr 47 tomograph ( siemens / cti ) . emission scans were initiated at 60,000 true counts per second after repeated intravenous bolus injections of doses of tracer with an activity of 500 mbq ( 13.5 mci ) . the tomography took place in a darkened room with participants ' eyes closed . the babble and running speech conditions the uptake lasted 90 seconds ( single frame ) at intervals of 10 min . after correction for scatter and measured attenuation , each pet frame was reconstructed with filtered back projection and smoothed with a postreconstruction 10 mm gaussian filter resulting in a resolution of 11 mm full - width - at - half - maximum ( fwhm ) . rules of regulation mean that participants who volunteer for scientific experiments may receive a total maximum radiation of 6 millisieverts ( msv ) within one year . here , the total radiation dose administered over the three times of scanning was approximately 5.58 msv . due to these restrictions , no preoperative baseline scans could be acquired . participants ' mr images were co - registered to an mr template averaged across 85 individual mr scans in talairach space , using a combination of linear and nonlinear transformations . each summed pet emission recording was linearly coregistered to the corresponding mr image using automated algorithms . to smooth the pet images for individual anatomical differences and variation in gyral anatomy , images were blurred with a gaussian filter resulting in final 14 mm at full - width half - maximum ( fwhm ) isotropic resolution . all images were processed using statistical parametric mapping 8 ( spm8 ; wellcome neuroimaging department , uk , ( http://www.fil.ion.ucl.ac.uk/spm/ ) ) . local maxima of activation clusters were identified using the montreal neurological institute ( mni ) coordinate system and then cross - referenced with a standard anatomical brain coordinate atlas . differences in global activity were controlled using proportional normalization ( gray matter average per volume ) . significance threshold for task main effects was set to p < 0.05 , family wise error ( fwe ) corrected for multiple comparisons . we tested the effect of side of implant and type of implant in a separate preanalysis . as we found neither main effects nor interactions with functional data involving these variables , we concluded that these factors had no significant effect on the results . the first analysis identified the main effects of time , speech / babble contrast , and history of hearing loss ( post hl versus pre hl ) and possible interactions between these effects in the whole brain , across ci participants . this analysis was performed as a single spm matrix in a factorial 3-way design with time , contrast , and subgroup as factors . to define a region - of - interest ( roi ) analysis 2 . the second analysis identified possible main effects of contrast , time , and interactions between these factors in the inferior frontal gyrus ( ifg ) , more specifically broca 's area ( ba 44/45 ) . this analysis was performed as two 2-way factorial analyses of the post hl and the pre hl subgroups separately . to define a region - of - interest ( roi ) , we created a mask based on bilateral inferior frontal gyri ( broca 's region ) , including the putative brodmann regions 44 , 45 , and 47 using the wfu pick - atlas . the third analysis investigated main effects of contrast and group ( ci versus nh ) at the ci baseline and possible interactions between these effects . this analysis was performed as a single spm matrix in a factorial 2-way design with condition and group as factors . to define a roi we found a main effect of speech / babble contrast across ci participants , regardless of subgroup , in bilateral superior temporal gyri ( f(1,78 ) = 60.14 ) . a t - test confirmed that the effect was driven by higher activity during running speech ( table 2 ; figure 1 ) . there was no significant main effect of time or group , nor any interaction between the effects . the roi analysis revealed significant interaction between the effects of contrast and group in ba 21/22 in the left superior temporal gyrus ( f(1,78 ) = 20.42 ) ( table 2 ; figure 2 ) . a plot of contrast estimates showed a difference between running speech and babble that was larger in the postlingual subgroup than in the prelingual subgroup ( figure 2 ) . in the bilateral ifg roi analysis , we found a main effect of speech / babble contrast in ba 47 in the postlingual subgroup . a t - test confirmed that the effect was driven by higher activity during running speech . furthermore , we found a main effect of time in left ifg ( broca 's area ba 45 ; f(2,60 ) = 14.19 ; p = 0.006 , fwe corrected ) ( figure 3 ) , with no significant interaction between contrast and time . the prelingual subgroup had no main effects in the bilateral ifg roi analysis ( table 3 ) . we found a main effect of speech / babble contrast across the ci and nh groups bilaterally in superior temporal gyri , in the left middle temporal gyrus , and in the right inferior parietal lobule . t - tests showed that the superior temporal gyri bilaterally and the left middle temporal gyrus were more active during running speech , while the right inferior parietal lobule was more active during babble . a t - test showed that this effect was due to higher activity of this area in the nh group than in the ci group . no interaction was found between the effect of contrast and the effect of group in whole - brain analysis ( table 4 ) . the roi analysis based on the main effect of contrast yielded a main effect of ci versus nh in secondary auditory cortex including ba 22 in the right superior temporal gyrus . a t - test showed that this effect was due to higher activity of this area in the nh group than in the ci group . furthermore , in the roi analysis , we found an interaction between the effect of speech / babble contrast and the effect of group in the right inferior parietal lobule ( table 4 ) . we found a significant effect of time in the post group ( (2,2 ) = 19.62 , p < 0.001 ) , but not in the pre group . post hoc tests showed that the effect was driven by the 51.3 percentage points endpoint gain ( mann - whitney u = 12.5 , p = 0.002 ) . furthermore , the post group scored significantly higher than the pre group at all three points of measurement ( bl : mann - whitney u = 0.000 , p = 0.004 ; mp : mann - whitney u = 0.000 , p = 0.004 ; ep : mann - whitney u = 0.000 , p = 0.004 ) . ceiling performance ( 100% correct ) was observed in all nh participants ( figure 4 ) . in this study , we aimed to examine the plastic changes which underlie the recovery of hearing after cochlear implantation . our results showed that across all ci recipients and all points of measurement , the bilateral middle and superior temporal gyri ( including , more specifically , brodmann areas 21 and 22 ) were significantly more active when participants listened to running speech than when they listened to multi - talker babble . thus , on average , the auditory brain regions , known to be involved in the processing of complex auditory stimuli [ 4951 ] displayed a clear distinction between speech - like noise and speech in recently implanted ci recipients . this confirms that both hemispheres are involved in the speech perception process , even during monaural auditory stimulation [ 29 , 36 , 52 , 53 ] . furthermore , the results showed a difference in the way ci recipients with postlingual hearing loss and recipients with prelingual hearing loss distinguish between speech and babble . the ci users with postlingual hearing loss displayed a greater activation during speech than during babble in ba 21 and 22 in the left superior temporal gyrus , indicating differential processing of the stimuli by the two subgroups . we speculate that the postlingually deaf listeners disengage attention when they are presented with the incomprehensible babble stimulus . in contrast , the prelingually deaf ci listeners may be equally attentive to the two stimuli , regardless of their nature , as reflected in undifferentiated activity . the postlingual subgroup not only possessed a moderate level of speech perception at baseline ( within 14 days after switch - on of the implant ) , but also made significant gains in performance , the majority of which occurred in the first three month period . in contrast , the prelingual subgroup had no baseline speech perception and only modest , if any , progress during the study period . this finding is consistent with expectation and implies an association between behavioral performance and brain activity related to the history of hearing loss . in prelingual deafness , the neuronal connections of the auditory pathways ( e.g. , measured as cortical auditory evoked potentials ) may not be established in the appropriate time window of opportunity [ 5456 ] . the subsequent electric stimulation at some time in adulthood may produce some hearing sensation , but the discrimination of sounds and time intervals remain defective [ 57 , 58 ] . the findings are compatible with naito et al . , who suggested that the reduced speech activation in prelingually deaf implant users could be explained by insufficient development of neuronal networks or their degeneration due to prolonged deafness . follow - up studies in the present population may provide interesting insight into the degree to which speech perception progresses in the prelingual subgroup in the long term . we found a main effect of time exclusively in broca 's area , and only in the postlingual subgroup . this is in line with recent studies showing that , in ci users , the activation of broca 's area during speech processing is negatively correlated with the duration of deafness and positively correlated with the progress in the restoration of speech comprehension [ 7 , 8 , 37 , 59 ] . this indicates that the changes in the auditory recovery process are most profoundly manifested in this specific area , which is associated with speech perception and production . this suggests that the area becomes increasingly activated , regardless of whether the stimulus makes semantic sense or not , or is active in the distinction between sense and nonsense . however , the absence of difference between speech and babble in broca 's area may also be explained by an increasingly high activity at rest in ci patients , as reported by strelnikov et al . . our whole - brain analysis revealed a significant activation of bilateral superior temporal gyri and left middle temporal gyrus during speech across ci participants at baseline and nh individuals . , who , in addition to bilateral superior temporal gyri , found significant speech activation in the right middle temporal gyrus , the left posterior inferior frontal gyrus ( broca 's area ) , and the left hippocampus in both normal participants and ci users . these further findings could be explained by the use of silence as contrast relative to the use of babble in the present study . interestingly , even though the nh participants received unilateral stimulation ( 6 right ; 6 left ) , the naito study found no significant difference in any brain area in either nh participants or ci users between right ear stimulation and left ear stimulation . the significant involvement of the right parietal lobule in the ci participants during babble suggests that , at this initial stage of the ci adaptation , ci listeners , unlike nh individuals , need to pay attention to the speech - like noise to determine its possible character . the observation that during speech stimulation the nh participants involved the caudate nucleus more than the ci participants may be explained by a reduction of the effort needed by the nh participants to deal with the well - known task of receiving a message . the caudate nucleus is a part of the striatum , which subserves among other tasks the learning of slowly modulated skills or habits . to the normal - hearing listener , the reception of auditory information is an every - day experience similar to following a known route ; for example , see wallentin et al . for a similar argument . in contrast , to ci listeners , auditory stimuli are nonhabitual in the strongest sense of the word , thus relying on other sources of processing . interestingly , naito et al . made a similar finding and speculated that though the caudate nucleus has been associated with various tasks ranging from sensorimotor tasks to pure thinking , the result may provide further evidence that the area has cognitive function and shows increased activity along with the increased activity in cortical language areas . in contrast to the significantly higher right - lateralized activation observed in nh individuals in the present study , giraud et al . showed a left - lateralized activation of temporal and frontal regions in nh controls . however , direct comparison between the two studies is difficult as there are several differences in study design and implant experience of the participants . found increased activity in right cerebellar cortex when running speech was comprehended relative to babble , but only in ci listeners with high speech comprehension . the authors speculated that this could be due to cognitive work of cerebellum subserving verbal working memory or a contribution of the right cerebellar hemisphere to precise representation of temporal information for phonetic processing . however , this finding was not replicated in the present study , which may reflect differences in duration of implant use and a mixture of speech comprehension levels . giraud and colleagues consistently demonstrated activation of areas ba 17/18 in the visual cortex when ci users responded to meaningful sounds . the authors argued that the process was associated with improvement of lip - reading proficiency , which is supported by findings in a behavioral study by rouger . a similar cross - modal interaction between vision and hearing was not replicated in the current study . the giraud study involved repetition of words and syllables and naming of environmental sounds , contrasted with noise bursts , as opposed to the current study , which involved passive listening to a story contrasted with speech - like noise . furthermore , sound was presented in free field , whereas in the current study , the auditory stimuli bypassed the microphones of the speech processor and were fed directly to the auxiliary input . finally , the strict conservative statistical methods used here preclude reporting of results that are not statistically significant when corrected for multiple comparisons . in scandinavia as in most european countries , cochlear implantation is administered by the public health care system and offered for free to all patients who meet the clinical implantation criteria ( < 40% open - set word - recognition scores ) . this includes patients with a prelingual hearing loss , despite recognition that these patients may have very limited linguistic benefit . as a result of this policy , the group of ci users in general is very heterogeneous , which was also the case in this study . while the difference in group size was far from optimal and may have confounded the results , we maintain that it is of high importance to also study cortical activity in prelingually deaf patients following cochlear implantation . in a previous study involving music training , we found that while little gain was achieved in speech perception , prelingual deafness did not preclude acquisition of some aspects of music perception . this implies that plastic changes take place also in the long - term deaf brain and that specific training measures could help these patients in achieving improved implant outcome [ 58 , 63 ] . our normal - hearing control group had a less optimal size , which may have made direct comparisons between groups less valid than desired . however , ethical restrictions limit the number of healthy participants in studies involving pet . as stated , the degree of deafness ( i.e. , residual hearing ) varied across subjects and may have influenced the adaptation to the implant . unfortunately , a correlation analysis between preoperative speech understanding and pet and behavioral results was not possible since such data were not available for all participants . the present pet study tested brain activation patterns in a group of recently implanted adult ci recipients and a group of normal - hearing controls , who listened to speech and nonspeech stimuli . ci listeners with postlingual hearing loss showed differential activation of left superior temporal gyrus during speech and speech - like stimuli , unlike ci listeners with prelingual hearing loss . this group difference was also reflected in a behavioral advantage for patients with postlingual hearing loss . furthermore , broca 's area was activated as an effect of time , but only in ci listeners with postlingual hearing loss . comparison of the ci listeners and the normal - hearing controls revealed significantly higher activation of the caudate nucleus in the normal - hearing listeners . the study demonstrates that processing of the information provided by the cochlear implant is highly related to the history of hearing loss . patients whose hearing loss occurred after the acquisition of language involve brain areas associated with speech comprehension , which is not the case for patients whose hearing loss occurred before the acquisition of language . finally , the findings confirm the key role of broca 's area in restoration of speech perception , but only in individuals in whom broca 's area has been active prior to the loss of hearing .

the most dramatic progress in the restoration of hearing takes place in the first months after cochlear implantation . to map the brain activity underlying this process , we used positron emission tomography at three time points : within 14 days , three months , and six months after switch - on . fifteen recently implanted adult implant recipients listened to running speech or speech - like noise in four sequential pet sessions at each milestone . ci listeners with postlingual hearing loss showed differential activation of left superior temporal gyrus during speech and speech - like stimuli , unlike ci listeners with prelingual hearing loss . furthermore , broca 's area was activated as an effect of time , but only in ci listeners with postlingual hearing loss . the study demonstrates that adaptation to the cochlear implant is highly related to the history of hearing loss . speech processing in patients whose hearing loss occurred after the acquisition of language involves brain areas associated with speech comprehension , which is not the case for patients whose hearing loss occurred before the acquisition of language . finally , the findings confirm the key role of broca 's area in restoration of speech perception , but only in individuals in whom broca 's area has been active prior to the loss of hearing .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

PMC3323861

heat intolerance has long been known to be a significant issue for people with ms , where an increase in core body temperature is associated with increased neurological symptoms [ 16 ] . people with ms rate high temperatures as one of the top three factors adversely affecting their symptoms , with stress and insufficient sleep being the other two . indeed , heat intolerance has a significant impact on the economic situation and quality of life of people with ms and their families , and managing heat - related problems is important to maintaining employment of people with ms . while neurological symptoms are frequently exacerbated by heat , these generally return to baseline when core temperature returns to normal , and colder temperatures usually reduce ms symptoms [ 911 ] . while it is clear that heat frequently exacerbates ms symptoms , patterns of relapse and exacerbation in relation to seasonal climate variations are variable and confounded by other factors such as viral infections and the modifying behaviours of people with ms , who routinely try to minimise their exposure to heat on hot days and nights [ 1214 ] . in australia , where the main climate variations range from temperate to tropical , residential air conditioning is widely used by people with ms to minimise the impact of hot days and nights on their symptoms . australian electricity prices are rising rapidly with annual increases of more than 10% annually between 2007 and 2010 . thus , in addition to the other economic difficulties faced by people with ms [ 16 , 17 ] , affordability of residential cooling is a major issue for many , frequently compounded by an early exit from the labour force and loss of income . in light of the above , ms australia perceived a potential need for government - funded financial assistance for residential electricity costs australians with ms on low incomes and sought relevant evidence on this issue to determine whether assistance was required and to underpin public policy advocacy and government lobbying efforts if government assistance was warranted . an extensive search of peer - reviewed literature and other sources indicated no studies of air - conditioning use by people with ms . the keeping cool survey consisted of nine questions covering the following topics ( see the supplementary material available online at doi:10.1155/2012/794310 ) : home air - conditioner use / nonuse and reasons why ; ambient temperature range when air conditioning is switched on ; age and type of air conditioner ( to estimate efficiency ) ; rooms of house that are air conditioned ; number of hours the air conditioner is used in particular months ; other home modifications to assist cooling ; the following question . as a person with ms , what happens when you get too hot ? i lack energy and require more rest . apart from fatigue , my other symptoms of ms become worse . i am unable to participate in normal social activities ( time with family or friends ) . i am unable to do my normal household duties ( e.g. , cleaning , cooking , etc . ) . demographic data for survey respondents was available from the australian ms longitudinal study ( amsls ) database , including sex , age , and postcode . of the respondents to the keeping cool survey , 66% ( 1,578 ) had also participated in one or both amsls economic surveys conducted in 2003 and 2007 . this , along with links to basic demographic information collected in other parts of the longitudinal survey , enabled additional analyses to be undertaken ( see section 2.3 below ) . the amsls is a nationwide , longitudinal cohort study described elsewhere [ 18 , 19 ] . briefly , the amsls maintains a large sample of volunteer participants from all australian states and territories ; 96% of whom have definite ms by the mcdonald diagnostic criteria , according to their neurologist or treating physician . the amsls project is approved by the act health human research ethics committee , an independent national health and medical research council - constituted human research ethics committee conforming to the ethical standards for human studies as per the 1964 declaration of helsinki . the survey was sent to 3,150 consenting participants of the amsls in september 2008 , of whom 2,385 responded ( 76% ) . analyses were undertaken comparing responses on all survey questions between those who would likely be eligible for government - funded assistance and those that would not . nonparametric tests were used to test categorical variables ( chi - square ) and ordinal data ( kruskal - wallis and mann - whitney ) . no p values approached significance , so all subsequent analysis considered the survey population as a whole . also , there were only three survey participants from the northern territory ; consequently their data were excluded from the analysis . economic modelling for estimating costs of air conditioner use by people with ms was undertaken utilising the survey results . this was necessary as it was found in piloting early versions of the questionnaire that it was not possible to get meaningful responses to direct questions about actual running costs . additionally , the bom created a new data set combining annual average maximum air temperatures and humidity into a single measure of apparent temperature ( a commonly used biometeorological measure ) , for this study . air - conditioner use at home by people with ms is a direct response to day - to - day weather conditions . one of the difficulties of examining climatic impacts on the use of air conditioners by people with ms is the climatic variation across time and place . additionally , air temperature data has significant limitations because moderate to high levels of humidity , coupled with hot days and nights , make it more difficult for people to keep cool . the bom routinely reports data on the number of hot and very hot days and nights , and national annual means for these are reported below in table 1 . australia has experienced an increased number of hot days and nights over time ( table 1 ) , which impacts on the air - conditioning needs of australians with ms and their associated costs . temperatures in 2008 when the survey was conducted were typical of the decade at 48.8 days 35c and 80.5 nights 20c . importantly , 35c is very hot for people with ms , as the survey results indicated that 29.9c is the mean external temperature at which australians with ms turn on their air conditioners . moreover , there are many more days over 30c than over 35c in most areas ; for example , across five weather stations in the sydney area there were 4.4 times more days 30c than there were 35c . table 2 presents the average maximum apparent temperature ( at ) data for australian states / territories and capital cities . at is an adjustment made to the ambient air temperature based on the level of humidity , which also impacts on personal comfort levels . the bom adjustments use absolute humidity with a dewpoint of 14c as the reference point ( with slight adjustments depending on the temperature ) . if the humidity is higher than the reference point , then the at is higher than the air temperature , and if the humidity is lower than the reference point , then the at is lower than the air temperature . in table 2 , at is ordered from highest to lowest , and ats for capital cities are included along with data for the states and territories within which at can vary by as much as 16c . the at for the capital cities may better represent the actual impacts on a state and territory populations . the demographic profile of survey participants was 79.0% female , and the mean age of the participants was 52 years , with a range of 2583 years . also for the 1,578 participants for whom economic data was available , 32.2% were likely to be eligible for a government - funded rebate on their electricity bills if a medical cooling rebate was in place . air conditioners were used by 81.9% of survey participants , with an additional 9.6% stating that they could not afford an air conditioner . in comparison , only 10.3% of respondents stated that they coped well in the heat and had no problems as a consequence of getting too hot . the most common issue was fatigue at 83.7% , followed by increases in symptoms other than fatigue at 61.9% . just under half of the respondents also reported reduced capacity for social , household and work activities , and self - care . small but notable proportions of people with ms also reported impacts on their use of medication ( 9.1% ) , doctor or other health professional visits ( 7.4% ) , and hospitalisation ( 3.4% ) . the mean national average external air temperature at which people turned on their air conditioners was 29.9c . tasmania was the state with the lowest mean at 26.4c , with the highest in south australia and western australia at 30.7c and 30.2c , respectively . figure 1 describes the imputed mean total hours of air - conditioner use per household across states and territories from september to april . imputed means were derived by using the midpoints from the categories selected by survey respondents : 0 = 0 ; 16 hrs = 3.5 hrs ; 712 hrs = 9.5 hrs ; 1318 hrs = 15.5 ; 1924 hrs = 21.5 . the types of air conditioners ( evaporative or heat exchanger ) used were almost identical to that of the general population , with heat exchangers dominating ( ranging from 60 to 98% ) and higher levels of evaporative more common in the driest areas ( western australia , south australia , victoria , and australian capital territory 3040% ) . newer air conditioners ( 03 years old ) were used in 36.2% of households , with 49-year - old units at 42.8% and those 10 years old or more at 20.9% . when the survey was conducted , inverter technology , which increases heat exchanger air conditioner efficiency by 2535% , was only present in those units 03 years old . nationally , 34.1% were cooling one room only , 32.7% were cooling two rooms , and 33.1% were cooling 4 rooms or more ( none stated that they were cooling 3 rooms ) . finally , respondents were also asked about their use of various measures to increase the thermal performance of their homes . nationally , 40.0% used external window coverings such as blinds and awnings , 80.5% used internal window coverings , 69.8% had ceiling insulation , 18.9% had roof vents , and 26.6% had wall insulation . while the figures for insulation are generally low by north american and european standards , ms households exceed national means for both wall ( 19% ) and ceiling ( 59% ) insulation . economic modelling was undertaken to convert the survey results into cost data ( see section 2.3 above ) . the first two sets , based on $ 0.15/kwh and $ 0.20/kwh , represent the range of prices across australia when the survey was conducted in 2008 . the other two sets of prices , $ 0.25 and $ 0.30 , represent the range of prices in 2011 . estimated nonevaporative air - conditioner cost data for households across australia in 2007 are included under the heading all . the present study has provided strong evidence that use of residential air conditioning by australians with ms is a major and common strategy for minimising the impact of a warm to hot climate . it also appears that people with ms in australia report a much higher incidence of heat sensitivity than those living in cooler climates . a recent survey in sweden found that only 58% reported heat sensitivity , compared to almost 90% in australia . this difference would appear to derive from climatic differences where australians are exposed to much higher temperatures ( see tables 1 and 2 ) consistently compared to their swedish counterparts where the temperature rarely exceeds 25c at the peak of summer . other research across a variety of locales has typically found a range of 6080% of people with ms self - reporting as heat sensitive in relation to symptoms increasing with exposure to heat . this further emphasises the importance of air conditioning for people with ms to keep cool in warmer climates such as australia . australian households that include people with ms are more likely than households nationally to ( a ) have an air conditioner , ( b ) use the air conditioner more often , and ( c ) incur the concomitantly higher electricity bills . these data are fundamental to the argument that government - funded financial assistance should be available to those on low incomes requiring medical - related cooling in the form of residential air conditioning . moreover , our results show that ms households had taken additional steps relative to other households to improve the thermal efficiency of their homes , above and beyond any government - funded contribution . the survey has therefore provided information for policy makers about the considerable problems people with ms incur with warm to hot weather , including regional variation in usage and need . nationally , people with ms spend between 4 and 12 times more on running their air conditioners than other households , with a national average of about 4 times more ( excluding evaporative air coolers ) . little research has been done to track household use of air conditioners generally , but one comparable survey ( based on recall and the completion of a questionnaire similar to the keeping cool survey ) performed in victoria in 2008 found that the average total hours of air conditioner use was 107 hours , compared to the present survey results in victoria of 1,508 hours . this indicates that ms households in victoria run their air conditioners by 14 times more hours annually than other households . for many households these additional costs must be met from relatively low incomes , with relatively high unemployment associated with ms . the high number of hours that ms households use their air conditioners relative to other households probably arises from several factors . people with ms are more likely to be at home as many are not working in paid employment , and they are more likely to be indoors during hot weather to escape the heat . the outside air temperature at which they turn on their air conditioners may also be at a lower threshold than other households given their heat sensitivity . while some of this higher use is probably slightly reduced through the higher levels of wall and ceiling insulation in ms households , these differences in thermal efficiency are relatively minor . strengths of the present study include the large national sample size ( almost 2,400 households with people with ms ) and the ability to subanalyse use of air conditioning by climatic and political regions . the latter enabled collation of data relevant to government policy makers in different australian states , leading to significant local outcomes in electricity subsidies for people with ms . limitations of the study include the use of retrospective questions on air - conditioning use , which may have introduced some recall bias , though in which direction remains unclear . the inability of households to identify the actual costs of running their air conditioners also necessitated economic modelling to obtain cost estimates . the sampling for the air - conditioning survey had some biases that are likely to have impacted on the results . previous examinations of the representativeness of participants in the amsls have found that the demographic characteristics of participants are similar to those of other recent studies , supporting the claim that the survey is broadly representative of australians with ms . nevertheless , there is likely an under - representation of those at the youngest and oldest ends of the age distribution and perhaps some bias towards over - representation of females . in australia people with ms use air conditioners extensively to keep cool at home when ambient external temperatures reach levels that begin to exacerbate their symptoms . the economic costs of this are increasingly problematic for low - income households , and the research results have been essential in demonstrating the need for government assistance to these households . subsequent campaigns by ms australia to obtain medical cooling rebates from several state governments have been successful , in part because of the presentation of such hard data to government policy makers . therefore , the use of patient self - reported data on salient life issues , facilitated by the existence of well - characterised national cohorts like the australian ms longitudinal study , can effectively be used for government lobbying to assist people with ms . finally , high levels of air - conditioner use by people with ms , and the need for assistance with electricity bills , are not the only policy implications of the survey . policy advocacy work also needs to be undertaken in relation to the replacement of old and inefficient air conditioners with more efficient units that have the capacity to cut operating costs substantially ( up to 5065% ) . also , given that peak demand for electricity in australia is during the afternoon and early evening of hot days , this is also when electricity supply overloads and blackouts are most likely to occur , making it essential for people with ms to have plans in place to minimise their exposure to heat when electricity is not available to their homes .

despite the known difficulties many people with ms have with high ambient temperatures , there are no reported studies of air conditioning use and ms . this study systematically examined air conditioner use by australians with ms . a short survey was sent to all participants in the australian ms longitudinal study cohort with a response rate of 76% ( n = 2,385 ) . questions included hours of air - conditioner use , areas cooled , type and age of equipment , and the personal effects of overheating . air conditioners were used by 81.9% of respondents , with an additional 9.6% who could not afford an air conditioner . regional and seasonal variation in air conditioning use was reported , with a national annual mean of 1,557 hours running time . 90.7% reported negative effects from overheating including increased fatigue , an increase in other ms symptoms , reduced household and social activities , and reduced work capacity . households that include people with ms spend between 4 and 12 times more on keeping cool than average australian households .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

PMC3203475

the temporomandibular joint ( tmj ) is a joint of the sinovial , double and bicondilar type . it is situated between the condylar process and the mandibular fossa of the temporal bone . it has an articular capsule , synovial membrane , ligaments and associated muscles . in comparison with other synovial joints , the tmj is unique because of the presence of the articular disc that separates it into two independent cavities , known as infra and supradiscal cavities , with distinct morphological features . at the end of the 12 gestational week morphologically , the articular disc is a small elliptic fibrocartilaginous plate shaped like an s in sagittal cut and having its bigger axis in the posteromedial direction . the articular disc is divided into four bands : anterior , intermediate , posterior and bilaminar zone . the disc also plays an important functional role in translation and rotation movements , in a broad and passive manner . the articular disc can be described as a concave lens with two faces ( superior and inferior ) , two margins ( medial and lateral ) and two extremities ( anterior e posterior ) . during the embryogenesis period , the articular disc of the tmj consists of a dense layer of mesenchymal cells that derived from the condylar blastema in the anterior region and from the glenoidal blastema in the posterior region . for sab , the architecture of the articular disc is complex , with collagen fibres predominating in the longitudinal direction of the superior and inferior faces and cross - sectional and interposed oblique fibres predominating in the intermediate portion . for minarelli and liberti , the disc has compact collagen bands in the intermediate band , with fibres running in the anteroposterior direction . takahashi and sato examined the distribution of types i and iii collagen , fibronectin and elastic fibres in the articular disc of 54 adult cadavers and 24 foetuses aged 12 , 16 , 20 , 24 , 28 and 32 weeks with light microscopy and scanning electron microscopy . according to the authors , at 12 weeks , the collagen fibres formed a thin layer on the entire disc ; at 16 weeks , the disc already had anteroposteriorly - oriented collagen fibres in the intermediate band and cross - sectional fibres were found in the most superficial layers of the disc ; finally , at 20 weeks , the authors found a disc consisting of a complex arrangement of collagen fibres , oriented in various directions . in terms of structural biology , the collagen fibres inside the articular disc of the tmj are composed mainly of type i collagen and small amounts of type iii collagen . according to takahashi and sato , the concentration of type - i collagens in the articular discs of foetuses aged 20 to 24 weeks increased in all bands , especially in the anterior and intermediate bands . meanwhile , the expression of type - three collagen in foetuses of the same gestational age increased only in the intermediate band . confirmed that a large number of fibroblast - like cells in the human tmj disc was co - localized with lumican expression in their specimens . in contrast , little lumican expression was observed in the vicinity of fibrochondrocytes and chondrocyte - like cells . matsumoto et al . demonstrated the presence of has3 in the chondrocyte - like cells of both normal and deformed tmj discs in adults . a study done at the institute of anatomy , university of catania , italy , used four articular discs from adult cadavers as controls . a multi - directional preservation of the collagen bands was found in all samples , in addition to a predominance of fibrocyte - like cells , and in a smaller aggregated amount , chondrocyte - like cells . the apparent presence of fibrocartilaginous areas also suggests the presence of type ii collagen fibers . fibrous proteins ( collagen and elastin ) , responsible for the shape and structure of the lamina propria , are associated with resistance and elasticity , while interstitial proteins ( glycoproteins and proteoglycans ) control viscosity , hydration , and the tissue volume extracellular matrix . according to gray et al . , the characteristics of the layers of the lamina propria are intimately associated with the arrangement and distribution of the fibrous proteins in the extracellular matrix , especially collagen fibres , which support and absorb the physical stress caused on the articular disc by joint movement . collagen belongs to a family of structural proteins with distinct chemical compositions , morphologies and functions . collagen is a component of many organs and tissues and is the main extracellular matrix element of multicellular animals , representing from 2530% of the protein mass of the organism . the different types of collagens have been organized into the following classes : fibrillar , associated with fibrils , non - fibrillar , filamentous , short- and long - chain . it has also been demonstrated that some types of collagen are capable of aggregating molecules to form heterotypical fibrils . the presence of heterotypical fibrils with different combinations in many tissues has already been reported . evidence of these combinations obtained with immunohistochemical analysis has been observed between types i and v collagen in human skin , tendon and cornea and between types ii and xi , and between i and ii in cartilage . given the scarcity of studies that focus morphologically on the types of collagen in the tmj disc of human foetuses , we believe a better structural knowledge of the relationship of these proteins with the articular disc of the tmj is necessary . thus , the objective of this study is to analyse the immunohistochemical expression of types i and iii collagen markers in the articular disc of the temporomandibular joint of human foetuses . the protocol of this study was submitted to the unifesp research ethics committee and approved under number 0225/05 . our sample consisted of 10 human foetuses supplied by the universidade federal de uberaba , resulting in 20 tmj with gestational ages from 17 to 24 weeks . the gestational age of the foetuses was determined by measuring the crown - rump ( cr ) length . all foetuses were preserved in a 10% formalin solution and dissected by removing the skin , subcutaneous tissue and exposing the deep structures . two sections of 3 m were taken from each paraffin block containing the collected material and were then spread on glass slides . the streptavidin - biotin technique was used to analyse the immunohistochemistry reactions of types i and iii collagens . sections were deparaffinised in three changes of xylene ( 10 min each ) and rehydrated in a graded series ( 100 , 80 and 70% ) of ethanol finishing in distilled water . antigen sites were retrieved by pretreatment with 0.5 m acetic acid for 90 min at 37c and multiple rinses with distilled water . endogenous peroxidases were quenched by incubating in 3% h2o2 ( 20 min , room temperature ) . the slides were then washed 32 min in pbs and incubated in milk powder solution for 20 min to block nonspecific binding of antibody . sections were incubated with 100 l of the primary antibody overnight at 4c.primary antibody was purified rabbit anti - human type i collagen , 1:800 ( novatec inc . , baltimore , md , usa ) and rabbit monoclonal anti - human type iii collagen ( novatec inc . ) . subsequently , slides were washed in pbs ( 32 min ) , and detection of the primary antibody was performed using the lsab kit ( dako cytomation , carpinteria , ca , usa ) . sections were incubated with biotinylated secondary antibody for 30 min , washed ( 32 min in pbs ) , and incubated with streptavidin - peroxidase ( hrp ) conjugate for 30 min . the sections were again washed ( 32 min in pbs ) before the antibody - hrp complex was visualized by incubation with diaminobenzidine ( dab lsab + system - hpr dako cytomation ) for 5 min . slides were briefly counter - stained in harris hematoxylin and dehydrated , and cover slips added . st louis , md , usa ) resin , analysed , and photographed with a light microscope ( olympus bx50 ) . the tissues recommended by the manufacturer were used as positive antibody controls.the negative controls were the same cases used as positive controls , which were submitted to the immunohistochemical reaction described above , except for the incubation with the primary antibody , done with a buffer solution . figure 1a shows numerous type i collagen fibres throughout the extension of the articular disc of the tmj . however , these fibres are more abundant in the posterior band of articular disc ( 1b ) . additionally , figure 1c shows a large concentration of type iii collagen fibres on the inferior face of the articular disc . figure 1d shows type iii collagen fibres in the intermediate band of the articular disc . figure 1immunohistochemical micrograph of type i collagen . a ) , foetus aged 19 weeks of intrauterine life ; there are collagen fibres in the entire disc ( a ) , mandibular head ( b ) and temporal bone ( c ) ; b ) , immunohistochemical photomicrograph of type i collagen in foetus at 22 weeks of intrauterine life ; the posterior band of the articular disc contains numerous complexly - arranged collagen fibres ; c ) , immunohistochemical micrograph of type iii collagen in foetus at 21 weeks of intrauterine life ; the inferior face ( a ) of the articular disc is delimited by numerous collagen fibres ; d ) , immunohistochemical micrograph of type iii collagen in foetus at 24 weeks of intrauterine life ; collagen fibres are arranged in many directions on the inferior face ( intermediate ) . the heart tissues used as positive controls for collagen i and iii are represented in panels e ) and f ) , respectively . a ) , foetus aged 19 weeks of intrauterine life ; there are collagen fibres in the entire disc ( a ) , mandibular head ( b ) and temporal bone ( c ) ; b ) , immunohistochemical photomicrograph of type i collagen in foetus at 22 weeks of intrauterine life ; the posterior band of the articular disc contains numerous complexly - arranged collagen fibres ; c ) , immunohistochemical micrograph of type iii collagen in foetus at 21 weeks of intrauterine life ; the inferior face ( a ) of the articular disc is delimited by numerous collagen fibres ; d ) , immunohistochemical micrograph of type iii collagen in foetus at 24 weeks of intrauterine life ; collagen fibres are arranged in many directions on the inferior face ( intermediate ) . the heart tissues used as positive controls for collagen i and iii are represented in panels e ) and f ) , respectively . minarelli and liberti have a stated that the anterior band contains 85% of type i collagen fibres and 15% of type iii collagen fibres , while the posterior contain 94% of type i collagen fibres and 6% of type iii collagen fibres . consider that the fibres inside the disc are composed mainly of type i collagen and are responsible for roughly 80% of the dry mass of the disc . figures 1a and 1b show numerous type i collagen fibres throughout the tmj disc , especially in the posterior band . figure 1c shows a great concentration of type iii collagen fibres located on the inferior face of the disc , while figure 1d shows complexly arranged type iii collagen fibres running in many different directions . this does not mean that the anterior , intermediate and posterior bands of the disc were not marked by the antibody , but when type i collagen fibres are compared with those of type iii , there is a prevalence of type i in the posterior band of the disc and a prevalence of type iii on the entire inferior face of the disc . in foetuses and children , type i collagen fibres have a stratified arrangement , organized in fibre bundles with an anteroposterior , laterolateral and oblique orientation forming mesh . in the posterior band of the disc , in agreement with our findings , the laterolateral fibre bundles were marked and , in agreement with minarelli and liberti , they formed a ring that surrounded the disc . in contrast , in the intermediate band of the disc , there was a prevalence of longitudinally oriented fibres in the anteroposterior direction . descriptions of the arrangement that constitute the architecture of the articular disc have been made in humans of different ages and also in primates . again regarding the composition of collagen fibres , it is important to point out that the aggregation of fibrillar collagen molecules to form heterotypical fibrils has already been described in many structures such as skin , tendon , meniscus , cartilage , human cornea and heart . in our study we observed that these heterotypical fibrils had probably formed , in agreement with takahashi and sato who demonstrated that the disc of foetuses of various gestational ages contains types i and iii collagens , and probably type ii , as judged berkovitz et al . by the presence of fibrocartilaginous areas in the articular disc . the distribution pattern of the fibres throughout the foetuses articular discs is different from that observed in adults articular discs . the following hypothesis may be considered : for the distribution patterns of types i and iii collagens to change as the foetus develops and in adults , some events , such as sucking , small mandibular movements and load , begin during the maturation stage , after the twelfth week of development , and are lifelong . thus , findings from this study may lead us to conclude that immunohistochemical expression of types i and iii antibodies is positive for the presence for these collagen fibres and heterotypical fibril meshes form between those two types of collagen .

the objective was to study the morphology of the articular disc and analyse the immunohistochemical expression of types i and iii collagen markers in the temporomandibular joint ( tmj ) disc of human foetuses of different gestational ages . twenty tmj from human foetuses supplied by universidade federal de uberaba with gestational ages from 17 to 24 weeks were studied . the gestational age of the foetuses was determined by measuring the crown - rump ( cr ) length . macroscopically , the foetuses were fixed in 10% formalin solution and dissected by removing the skin and subcutaneous tissue and exposing the deep structures . immunohistochemical markers of type i and iii were used to characterize the existence of collagen fibres . analysis of the immunohistochemical markers of types i and iii collagen revealed the presence of heterotypical fibril networks .

Introduction Materials and Methods Results Discussion

PMC2896150

the variety of biochemical functions that are being carried out by rna molecules is mesmerizing . many rnas such as ribosomal rna , rnaase p or trna attain a defined secondary and tertiary structure that is vital to their function . experimentally determined structures are only available for a small fraction of rnas that are of interest . this makes the computational prediction of the base - pairing pattern ( the secondary structure ) of rna an important problem . one major breakthrough was the development of dynamic programming algorithms that could predict the minimum free energy secondary structure of rna sequence assuming that the structures are non - nested ( 15 ) . subsequently , dynamic programming algorithms have been extended to allow certain classes of pseudoknots ( 6,7 ) . many rna secondary structure prediction algorithms ( including the one presented here ) are based on the idea of iteratively adding substructures to an initially unfolded sequence ( 8,9 ) . genetic algorithms are an example of such algorithms and have proven very useful for exploring pseudoknotted structures and sub - optimal rna structures ( 1014 ) . allowing pseudoknots is desirable simply because rna structures determined by x - ray crystallography or nmr revealed that many rnas contain non - nested base pairing interactions . allowing all possible base pairing interactions leads to the potential problem for structure prediction approaches that not only are there many more conformations to consider , but also many conformations are not sterically feasible . here , we describe a computational approach for rna secondary structure prediction that has no restriction in terms of pseudoknot complexity , but additionally checks the steric feasibility of the considered conformations . the described approach of rna secondary structure prediction is based on the idea of maximizing matching helices in a secondary structure ( 10 ) . a flow chart of the algorithm is shown in figure 1 . briefly , the method works as follows : initially , a list ( called a stem - list ) of all possible helices with more than 3 bp is generated . the secondary structure prediction is performed by picking the best - scoring structure obtained after 50 folding simulation runs . the score is set to be the sum of the free energy contribution of the already placed helices . each folding simulation run is performed by picking helices from the stem list with a boltzmann - weighted probability . estimating the free energy contribution of an rna double - helix is accomplished using the rna vienna package ( 2 ) . each chosen helix is represented by a very coarse - grained 3d representation in a virtual 3d workspace . an rna double helix is represented by a cylinder ( using a radius of 6.5 and a length of 2.7 times the number of base pairs ) that is capped with a half - sphere on both ends . this shape is called a capsule . a schematic diagram of the mapping of an rna secondary structure into a highly coarse - grained 3d representation is shown in figure 2 . the main reason for choosing capped cylinders over regular cylinders is the computational efficiency of collision detection . single stranded regions between helices are represented as constraints for the maximum distance between the ends of the capped cylinders . a newly chosen capped cylinder is placed into the 3d simulation space at a random position such that the distance - constraints are fulfilled . the distance constraints are a function of the single - stranded sequence lengths between connected helices . the maximum distance between helix ends is 2.0 + n*8.0 with n being the sequence separation . figure 2.scheme for mapping between an rna secondary structure ( a ) and the used 3d coarse - grained representation ( b ) . only those rna secondary structures can be a ( partial ) solution of a secondary structure prediction , for which the algorithm succeeds in placing the corresponding capped cylinders such that they do not collide and do not violate distance constraints . a flow - chart depicting the algorithm for predicting rna secondary structures . scheme for mapping between an rna secondary structure ( a ) and the used 3d coarse - grained representation ( b ) . only those rna secondary structures can be a ( partial ) solution of a secondary structure prediction , for which the algorithm succeeds in placing the corresponding capped cylinders such that they do not collide and do not violate distance constraints . if cylinders collide , the newly placed capped cylinder is placed at a different random position . if after 20 attempts the newly placed capped cylinder is still colliding with previously placed capped cylinders , the positions of all capped cylinders are optimized in order to minimize collisions and constraint violations . if no collision - free position can be found , the newly chosen helix and its capped cylinder representation is discarded . helices that are part of the stem - list and that share bases with the newly placed helix are removed from the stem - list . in the next iteration the next helix is chosen until no more helices can be placed . once no more fifty simulation runs are performed and the overall best - scoring structure is returned to the user . the web server has been implemented using the grails framework ( 18 ) , which is based on the groovy programming language . for a secondary structure prediction request , the web server launches the cylofold binary on a linux compute cluster . after the prediction result has been generated , the program varna ( 19 ) a user of the cylofold prediction web server can start a secondary structure prediction request by entering ( pasting ) a nucleotide sequence ( as raw characters or in fasta form , both acgu and acgt alphabets are accepted ) into the web form and pressing submit. the maximum sequence length that is currently accepted by the web server is 300 nt . the initial return of the web server is a unique i d , which is needed if one wants to access results at a later time . due to the compute - intensive approach for the prediction , it can take several minutes for the server to finalize a secondary structure prediction . the user can access the results by one of three methods : a simple reload of the initial result page will update the status of the prediction and will eventually contain the prediction results . alternatively , the user can bookmark the initial result page in the web browser and return to it at a later time . lastly , the unique i d provided after submitting the secondary structure prediction compute request can be used to access the results using another web form available on the server home page . a typical output from a completed rna structure prediction the prediction result is presented to the user in three different formats : ( i ) an image of the predicted rna secondary structure created by varna ( 19 ) ; ( ii ) an extended bracket notation in which nested base pairs are denoted as pairs of nested parentheses and helices corresponding to pseudoknot interactions are denoted as letters ; ( iii ) the ct file format that is also generated by other programs such as mfold ( 5 ) . this format contains a list of the indices of the bases and their predicted base - pairing partners . the shown sequence corresponds to the bacteriophage t2 gene 32 mrna pseudoknot ( pdb 2tpk ) . the shown sequence corresponds to the bacteriophage t2 gene 32 mrna pseudoknot ( pdb 2tpk ) . a user of the cylofold prediction web server can start a secondary structure prediction request by entering ( pasting ) a nucleotide sequence ( as raw characters or in fasta form , both acgu and acgt alphabets are accepted ) into the web form and pressing submit. the maximum sequence length that is currently accepted by the web server is 300 nt . the initial return of the web server is a unique i d , which is needed if one wants to access results at a later time . due to the compute - intensive approach for the prediction , it can take several minutes for the server to finalize a secondary structure prediction . the user can access the results by one of three methods : a simple reload of the initial result page will update the status of the prediction and will eventually contain the prediction results . alternatively , the user can bookmark the initial result page in the web browser and return to it at a later time . lastly , the unique i d provided after submitting the secondary structure prediction compute request can be used to access the results using another web form available on the server home page . a typical output from a completed rna structure prediction the prediction result is presented to the user in three different formats : ( i ) an image of the predicted rna secondary structure created by varna ( 19 ) ; ( ii ) an extended bracket notation in which nested base pairs are denoted as pairs of nested parentheses and helices corresponding to pseudoknot interactions are denoted as letters ; ( iii ) the ct file format that is also generated by other programs such as mfold ( 5 ) . this format contains a list of the indices of the bases and their predicted base - pairing partners . the shown sequence corresponds to the bacteriophage t2 gene 32 mrna pseudoknot ( pdb 2tpk ) . the shown sequence corresponds to the bacteriophage t2 gene 32 mrna pseudoknot ( pdb 2tpk ) . the performance of the new rna secondary structure prediction method was evaluated using two different data sets . data set 1 ( corresponding to the results shown in table 1 ) consists of 26 rna sequences , whose tertiary structure is available in the protein data bank ( pdb ) . the reference secondary structure was obtained by extracting the base pair information from the pdb coordinate file using the program rnaview ( 20 ) . data set 2 consists of 241 rna sequences and secondary structures originating from pseudobase ( 21,22 ) . table 1.prediction results corresponding to 26 rna structures that are available in the protein dank bankpdbdescriptionlpkfcfpkhkufmccsnsppvmccsnsppvmccsnsppvmccsnsppv1a60tymv trna - like structure4413.60.740.770.710.961.000.930.830.770.910.830.770.911cx0hdv ribozyme7222.20.010.000.000.010.000.000.010.000.000.010.000.001e95srv-1 pseudoknot3633.31.001.001.001.001.001.001.001.001.000.700.501.001hvuhiv rt bind . intron700.00.910.870.950.810.830.790.810.830.790.960.960.961l2xviral rna pseudoknot2722.20.941.000.890.941.000.890.790.631.000.790.631.001q9a23s rrna sarcin / ricin270.00.910.831.000.770.830.710.861.000.750.770.830.711u8dguanine riboswitch6711.90.870.870.870.880.781.000.880.781.000.880.781.002a43luteoviral pseudoknot2623.00.931.000.880.931.000.880.750.571.000.750.571.002g1wtmrna pseudoknot2218.10.811.000.670.861.000.750.810.671.000.810.671.002gissam- riboswitch948.50.800.760.850.800.760.850.860.860.860.550.550.552hoothi - box riboswitch830.00.700.670.740.580.620.540.580.620.540.580.620.542k95p2b - p3 telo - merase rna4837.50.890.801.000.890.801.000.750.80.710.540.400.752oiul1 ribozyme ligase adduct710.00.860.780.950.980.961.000.980.961.000.981.001.002qushammerhead ribozyme682.90.950.911.000.950.911.000.950.911.000.951.001.002qwysam - ii riboswitch5226.90.480.460.500.480.460.50.340.310.40.350.310.402rp0pemv1 mrna pseudoknot2615.30.881.000.780.881.000.780.840.711.000.840.711.002tpkt2 gene 32 mrna p.k.3627.71.001.001.001.001.001.000.710.580.880.630.580.70361ddomain e of 5s rrna190.00.861.000.750.861.000.750.830.830.830.830.830.833diglysine riboswitch17330.10.890.850.930.740.720.760.740.720.760.740.720.763fu2class - i preq1 riboswitch3218.80.790.631.000.790.631.000.790.631.000.790.631.003phptymv p.k . frame - shifting p.k.2722.20.941.000.890.941.000.890.790.631.000.790.631.00meanall0.830.850.830.820.840.810.750.710.830.730.650.83meanno pseudoknots<5.00.870.850.890.810.840.800.820.840.810.870.900.87meanpseudoknots>5.00.810.840.800.820.840.820.730.650.840.660.550.80l , sequence length ; pkf , fraction of pseudoknot interactions ; for each of the four different prediction methods ( cf , cylofold ; pk , pknotsrg ; hk , hotknots 2.0 ; uf , unafold ) we report three different measures of prediction quality ( sns , sensitivity ; ppv , positive predictive value ) . prediction results corresponding to 26 rna structures that are available in the protein dank bank l , sequence length ; pkf , fraction of pseudoknot interactions ; for each of the four different prediction methods ( cf , cylofold ; pk , pknotsrg ; hk , hotknots 2.0 ; uf , unafold ) we report three different measures of prediction quality ( sns , sensitivity ; ppv , positive predictive value ) . in order to quantify the time - complexity of the folding method , we fitted a function of the form a*n ( with n being the number of residues in the input sequence ) to the execution time needed for the cases of the 241 sequence set . we found that the execution time ( measured in seconds ) of the structure prediction is well described by the function 2.74 * 10*n . the timing evaluation was performed on a computer with 4 gb of ram and an intel 64-bit xeon processor ( 3.0mhz ) . we report in tables 1 and 2 prediction results for these two data sets together with the corresponding results obtained by running the rna secondary structure prediction programs hotknots 2.0 ( 8) , pknotsrg ( 7 ) and unafold ( 23 ) . table 2.prediction results for a set of 241 rna sequences that are part of pseudobase for the programs cylofold , pknotsrg ( 7 ) , hotknots 2.0 ( 8) and unafold ( 23)mccsnsppvcylofold0.7520.7630.747pknotsrg0.7480.7530.756hotknots 2.00.6110.5650.684unafold0.5970.5320.692sns , sensitivity of predicted base pairs ; ppv , positive predictive value . prediction results for a set of 241 rna sequences that are part of pseudobase for the programs cylofold , pknotsrg ( 7 ) , hotknots 2.0 ( 8) and unafold ( 23 ) sns , sensitivity of predicted base pairs ; ppv , positive predictive value . the average matthews correlation coefficient ( mcc ) obtained by comparing the base pairing pattern of the predicted secondary structures with their respective reference secondary structure is for data set 1 and cylofold 0.83 ; this can be compared to pknotsrg ( 0.82 ) , hotknots 2.0 ( 0.75 ) and unafold ( 0.73 ) ( see row of table 1 named we divided this data set into two subsets according to the fraction of pseudoknot base pairs in the respective structures . the eight pdb structures with < 5% pseudoknotted base pairs correspond to an average mcc of 0.87 for cylofold compared to 0.81 for pknotsrg , 0.82 for hotknots 2.0 and 0.87 for unafold . the 18 structures listed in table 1 that have a pseudoknot amount > 5% correspond to an average mcc of 0.81 for cylofold , 0.82 for pknotsrg , 0.73 for hotknots 2.0 and 0.66 for unafold . using the larger data set 2 , one obtains an average mcc of 0.752 for cylofold and 0.748 for pknotsrg one can see the rna secondary structure predictions obtained by cylofold correspond to the highest mcc ( compared to the programs pknotsrg , hotknots 2.0 and unafold ) . it also has the highest average base pair prediction sensitivity ( 0.763 ) . for another measure , the positive predictive value ( how often are predicted base pairs part of the reference secondary structure ) , all programs obtain averages between 0.68 and 0.76 for data set 2 with pknotsrg leading with a value of 0.756 . it should be noted that the mcc is often used as an overall measure of prediction quality , while sensitivity , specificity and positive predictive value capture certain other aspects of the prediction quality . the key advantage of cylofold is that there is no restriction in terms of the classes of pseudoknots that are being considered . also , it should be noted that the employed model of simulated rna folding by placing helices with a probability according to their free energy contribution is in essence very simple ( 24 ) . in that sense it is surprising how well the method performs , and it should be an encouragement to continue to develop rna folding algorithms that are substantially different from established approaches . we show using two different data sets that the prediction accuracy ( mcc ) is comparable to the rna secondary structure prediction program pknotsrg . another novel aspect is that at each step during the simulated folding process , the steric feasibility of the predicted structures is checked for steric feasibility using a highly coarse - grained 3d representation . the method is made available in the form of a user - friendly web server . this project has been funded in whole or in part with federal funds from the national cancer institute , national institutes of health , under contract hhsn26120080001e . this research was supported by the intramural research program of the nih , national cancer institute , center for cancer research .

computational rna secondary structure prediction approaches differ by the way rna pseudoknot interactions are handled . for reasons of computational efficiency , most approaches only allow a limited class of pseudoknot interactions or are not considering them at all . here we present a computational method for rna secondary structure prediction that is not restricted in terms of pseudoknot complexity . the approach is based on simulating a folding process in a coarse - grained manner by choosing helices based on established energy rules . the steric feasibility of the chosen set of helices is checked during the folding process using a highly coarse - grained 3d model of the rna structures . using two data sets of 26 and 241 rna sequences we find that this approach is competitive compared to the existing rna secondary structure prediction programs pknotsrg , hotknots and unafold . the key advantages of the new method are that there is no algorithmic restriction in terms of pseudoknot complexity and a test is made for steric feasibility . availability : the program is available as web server at the site : http://cylofold.abcc.ncifcrf.gov .

INTRODUCTION ALGORITHM IMPLEMENTATION Usage RESULTS AND DISCUSSION CONCLUSION FUNDING

PMC4349040

wrd wielu problemw sygnalizowanych przez dorastajcych pacjentw z wrodzonymi wadami serca ( grown - up congenital heart przekonanie o bliej nieokrelonym ograniczeniu oglnej sprawnoci zwizanym z wad serca i jej wczeniejszym leczeniem . poprawie aktywnoci fizycznej pacjentw z guch moe suy odpowiednio przygotowany program kompleksowej rehabilitacji kardiologicznej ( krk - guch ) stanowicy kolejny , odlegy etap leczenia . ocena wpywu krk - guch na aktywno fizyczn pacjentw w odlegym okresie po chirurgicznej korekcji wrodzonych wad serca . do badania wczono 57 pacjentw ( 30 kobiet , 27 mczyzn ) w wieku 23,7 4,1 roku , bdcych minimum 12 miesicy po korekcji operacji ubytku w przegrodzie midzykomorowej ( ventricular septal defects vsd ) lub w przegrodzie midzyprzedsionkowej ( atrial septal defects asd ) . grupa a ( n = 31 ) , oraz nieuczestniczc w programie rehabilitacji grupa b ( n = 26 ) . u wszystkich pacjentw wykonano wstpne badania czynnociowe , po czym wdroono program krk - guch . po 30 dniach od badania wstpnego ponownie oceniono pacjentw z obu grup , stosujc takie same narzdzia badawcze jak we wstpnym badaniu . u pacjentw z grupy nierehabilitowanej ( grupa b ) stwierdzono istotnie nisz aktywno fizyczn po miesicu od badania wstpnego ni w grupie pacjentw rehabilitowanych w ramach programu krk - guch ( grupa a ) . i tym samym najprawdopodobniej jako ycia pacjentw w pnym okresie po chirurgicznej korekcji wrodzonych wad serca . zaproponowany program krk - guch moe stanowi uzupenienie holistycznej opieki w opisywanej grupie pacjentw po korekcji wrodzonych wad serca . adolescents and young adults after surgery of grown - up congenital heart ( guch ) defects constitute a growing group of patients requiring cardiac care and multifaceted support . the reduction of early mortality and the prolongation of survival among patients with congenital heart defects ( chds ) may appear as a tangible success [ 1 , 2 ] . however , apart from good short - term results and long - term survival , to fully assess the efficacy of the interventional treatment of cardiac defects , a broadly understood quality of life evaluation of adolescent patients with cardiac issues that were successfully treated during childhood is needed . among the many issues raised by adolescent guch patients , the widespread belief concerning unclearly defined restrictions in general fitness , associated with congenital heart defects and their previous treatment , appears to be fundamental . it directly impacts the patients everyday life and reduces their physical activity . undergoing cardiac surgery in childhood leads to a decrease in expectations , which naturally reduces the readiness of the patients themselves to take up various forms of physical activity . notwithstanding , the physical activity of guch patients may be improved by a properly prepared comprehensive cardiac rehabilitation ( ccr - guch ) program , serving as another stage of treatment with proven influence on exercise tolerance and quality of life . the present study evaluates the impact of comprehensive cardiac rehabilitation on the physical activity of guch patients participating in a specially developed authorial program dedicated to this group of patients . its basic premise is that the low physical activity of guch patients increases their predisposition to cardiovascular diseases proportionally more in the case of those less active , similarly to the general population . the issue appeared especially interesting due to the potential for increasing the activity of adolescent patients with congenital heart diseases , which could consequently improve their general condition and reduce the risk of additional cardiovascular problems natural for this age in long - term observation . the aim of this study was to evaluate the influence of the ccr - guch program on the long - term physical activity of adolescent patients after chd surgery . fifty - seven patients with chds ( 30 women and 27 men ) at the mean age of 23.7 4.1 years , who met the inclusion criteria and provided their free and informed consent , were invited to participate in the program of comprehensive cardiac rehabilitation designed at the chair and clinic of rehabilitation of the medical university of gdask in the years 2007 - 2009 . all the patients had undergone cardiac surgery procedures in childhood ( at the age of 6 on average ) ; these included either the correction of ventricular septal defects ( vsd ) or the repair of atrial septal defects ( asd ) . out of all patients invited to participate in the ccr - guch program , 31 patients ( 17 women and 14 men ) were ultimately included in the program . the remaining 26 patients , who did not participate in the rehabilitation program due to logistic , economic , or social reasons , were provided information about the program as well as about the expected positive results of the involved controlled physical exertion and its importance for normal functioning . the chd patients constituting the study group were divided into two subgroups : those participating in rehabilitation ( a ) and non - participants ( b ) . the control group consisted of 30 healthy students ( 15 women and 15 men ) at the mean age of 24.4 1.97 years , free of any additional comorbidities , or physical / mental limitations , who provided their free and informed consent for their participation and the anonymous use of the obtained results for the purpose of the present study ( group c ) . identical inclusion criteria were used for the examination of guch patients ( groups a and b ) : clinical diagnosis condition after the repair of shunt - related cardiac defects ( asd or vsd ) employing median sternotomy ; time from the cardiac surgery over 12 months . the inclusion criteria for all three groups ( a , b , and c ) were : age over 18 years as well as free and informed written consent for participation in the study . the exclusion criteria included : active inflammatory diseases , life - threatening cardiac dysrhythmias , impaired motor function preventing the patients from performing the tasks of the rehabilitation program , coexistence of other heart defects ( congenital or acquired ) , significant deterioration of clinical condition within the past month , acute cardiac or neurological events within the past 6 months , positive results of the cardiac diagnostic test , lack of consent , or mental illnesses precluding patient cooperation . no statistically significant differences between groups a and b were observed in terms of the distribution of sex , height , body mass , or the basic functional indicators of the circulatory system . the mean age of the patients at the time of surgery was 5.18 2.8 years . the resting heart rate ( hrrest ) was 90 bpm on average ; right bundle branch block ( rbbb ) was revealed on resting electrocardiograms of 15 patients , premature ventricular contractions ( pvc ) were diagnosed in 4 patients , and premature supraventricular contractions ( psvc ) were diagnosed in 2 patients . no statistically significant differences were noted between groups a , b , and c in terms of selected initial anthropometric indices ( table i ) . patient characteristics for the study groups ( a undergoing rehabilitation , b no rehabilitation ) and controls ( c ) bmi body mass index , sd standard deviation all patients ( groups a , b , and c ) underwent physical examination , and their medical histories were obtained . the patients underwent cardiopulmonary exercise testing on a cycloergometer using the metasoft studio software ( cortex ) ; the selected ramp protocol consisted of an initial load of 20 w increasing by 10 w per minute . limits of maximal fatigue were put in place along with the standard indications for test termination ; the following parameters were evaluated : hrrest , hrmax , exercise time , exercise load in watts , vo2 peak . the evaluation of physical activity was based on the stanford questionnaire , which served as a tool for the objective assessment of patients with different parameters . it was composed of questions concerning the patients current lifestyle and was divided into two parts , including questions about everyday exercise of low - activity ( stanford i ) and high - activity exercise ( stanford ii ) . each test item was worth one point , and the final score reflected the current physical activity of the respondents . additionally , a written questionnaire was drawn up especially for the purposes of the ccr - guch program . the subjects from both study groups ( a , b ) and the controls ( c ) were asked to answer questions about their lifestyle ( whether they considered themselves physically active and whether they were apprehensive of engaging in some forms of physical activity ) . they were also asked about their participation in physical education classes during their school years and about tobacco use . the guch patients ( a , b ) were also asked whether they had ever participated in cardiac rehabilitation and whether they saw the need for engaging in controlled physical training sessions . after 30 days following the initial examination and after completion of the ccr - guch program , the physical activity of patients in groups a and b was evaluated again , using the same methods as during the initial examination ( stanford i and ii questionnaires , own questionnaire ) . the ccr - guch program was implemented during the course of four weeks and was based on recommendations concerning comprehensive cardiac rehabilitation in adults . the kinesiatric program involved a cycle of half - hour monitored cycloergometer training sessions and general fitness training with elements of aerobics and nordic walking . at the gym , the subjects engaged in exercises with and without equipment as well as resistance exercise . breathing exercises were introduced in order to strengthen the respiratory muscles and to teach proper breathing both during exertion and in situ ations requiring the improvement of ventilation , e.g. dyspnea or fatigue . resistance training was performed 2 - 3 times per week on fitness stations using weights up to 20 kg ; the sessions consisted of no more than 4 series of 15 repetitions . the psychological influence consisted of emotional support , the stimulation of self - confidence , and the improvement of self - esteem . during the educational part of the program , which included classes conducted by a cardiologist , a psychologist , and a dietician , the patients learned methods of coping with emotional stress and changing the habits adversely affecting their predisposition to cardiovascular diseases . the patients were instructed how to measure their pulse ( monitoring the pulse and assessing potential cardiac dysrhythmias ) and arterial pressure . moreover , the participants were instructed in the proper use of everyday exercise , including proper respiration during various forms of activity ( walking , running , resistance training ) . they also received advice concerning the optimal selection , planning , and performance of exercises and were shown various forms of physical activity , including sports and recreational activities which exert favorable effects on the cardiovascular system and facilitate the achievement of good short - term and long - term results [ 8 , 9 ] . the performed analysis of the initial stanford questionnaire results demonstrated that the guch patients ( a and b ) were less physically active than their healthy peers ( mean scores : stanford i 1.9 vs. 2.9 points , p < 0.001 ; stanford ii preliminary results of the physical activity examination among the study groups guch patients ( a and b ) and the controls healthy students ( c ) before the start of the ccr - guch program guch grown - up congenital heart , ccr comprehensive cardiac rehabilitation , stanford i low - activity exercise , stanford ii high - activity exercise subsequently , the guch patients undergoing rehabilitation ( a ) were compared with the group that did not undergo the ccr - guch program ( b ) . both groups were characterized by similar baseline physical activity according to the initial stanford questionnaire ( table iii ) . results of the stanford questionnaire among the study groups ( a undergoing rehabilitation and b no rehabilitation ) before the start of the ccr - guch program ccr comprehensive cardiac rehabilitation , guch grown - up congenital heart , stanford i low - activity exercise , stanford ii high - activity exercise forty - seven guch patients ( 82% ) did not participate , while all students ( 100% ) did participate in physical education during their school years . apprehension concerning engaging in physical exertion in everyday life was reported by 35 guch respondents ( 61% ) , while all students declared that they felt no fear of exercise . twenty - six guch patients ( 45.6% ) declared themselves to be physically active in everyday life ; all the students answered this question in the same manner . all the guch patients ( 100% ) declared the need to improve their physical capacity and activity . similar answers were given to the question concerning the readiness to participate in controlled ccr - guch training sessions ; however , 26 patients ( 45.6% ) did not ultimately participate in the program . three of the guch patients ( 5.2% ) and 2 students ( 6.4% ) reported tobacco use . after the intensive rehabilitation program conducted over the course of 1 month , the subjects were again asked to fill out the stanford questionnaire . seven patients who did not undergo rehabilitation ( group b ) did not answer the questions . the analysis of the remaining 50 questionnaires demonstrated that , after the period of 1 month , the patients who were not included in the ccr - guch program ( group b ) were characterized by significantly lower physical activity in comparison to the patients participating in the training sessions ( group a ) ( mean scores : stanford i 1.8 vs. 3.2 points , p < 0.001 ; stanford ii 0.13 vs. 1.2 points , results of the stanford questionnaire among the study groups ( a undergoing rehabilitation and b no rehabilitation ) after the end of the one - month ccr - guch program ccr comprehensive cardiac rehabilitation , guch grown - up congenital heart , stanford i low - activity exercise , stanford ii high - activity exercise the analysis of both parts of the questionnaire also revealed that , after the completion of the ccr - guch program , the 31 patients from group a engaged in both low - activity ( stanford i ) and high - activity exercise ( stanford ii ) much more frequently ( p < 0.001 and p < 0.001 ) ( fig . the results of the stanford questionnaire in group a before the start and after the completion of the ccr - guch program are presented in table v. results of the stanford questionnaire among the study groups ( a undergoing rehabilitation and b no rehabilitation ) before the start and after the end of the one - month of the ccr - guch program ; a ) stanford i ; b ) stanford ii results of the stanford questionnaire in study group a ( undergoing rehabilitation ) before the start and after the end of the ccr - guch program ( n = 31 ) ccr comprehensive cardiac rehabilitation , guch grown - up congenital heart , stanford i low - activity exercise , stanford ii high - activity exercise the unequivocally positive opinion of the effects of the ccr - guch program , expressed by 24% of the guch patients , should be underscored at this point . asked about the benefits that they believed were achieved by participating in the program , 20 group a patients ( 64.5% ) responded that their subjective well - being was much better , 5 patients ( 16.1% ) wrote that they tired less during everyday activities , 2 patients ( 6.4% ) no longer feared increasing the load of physical exercise , and 3 patients ( 9.6% ) felt safer during their everyday activity . their descriptive answers to the questionnaire handed out after the completion of the rehabilitation program were more likely to include information that they tended to use the stairs more often than the elevator , that they tended to walk rather than drive for short distances , and that they would more frequently go out for walks after dinner or supper . the questionnaire score reflecting improvements in everyday physical activity corresponds with the results of the stanford i questionnaire , assessing the same type of activity : the mean stanford i score changed from 1.97 points before ccr - guch to 3.2 after completion of the program . congenital heart defects surgery is typically performed during the patient 's childhood ; the age of patients undergoing these operations is increasingly lower , and increasingly younger children undergo successful cardiac surgical treatment using state of the art technology . the dominant group among adolescents and young adults with chds is formed by those who had undergone cardiac surgery , but , unfortunately , have predominantly sedentary lifestyles or undertake exercise that is not recommended for their health condition . this often results from the lack of precise information concerning the type and intensity of exercise that they could engage in . telling these patients : you can do everything , but in moderation or the basis for the selection of physical activity for individuals with chd is constituted by the standards of sports cardiology included in the recommendations of consecutive sports conferences in bethesda . the newest recommendations concerning the use of physical exercise by individuals with chd were listed in the european society of cardiology guidelines for the management of grown - up congenital heart disease ( new version 2010 ) . regrettably , many adolescents and young adults after the surgical treatment of simple shunt - related cardiac defects avoid intense physical activity [ 13 , 14 ] . the bethesda recommendations permit any kind of exercise after the successful and uneventful repair of simple intracardiac shunts . the guidelines of the european society of cardiology ( esc ) also contain no contraindications for physical exercise in this patient group . the decided majority of patients operated on due to simple shunt - related heart defects ( atrial and ventricular septal defects ) may perform physical exercise of any kind and intensity starting from the 6th postoperative month , provided no complications , pulmonary hypertension , or dysrhythmias are observed . it should be stressed that information concerning the physical exercise recommended to this patient group is still lacking ; as a result , many patients are unsure what types of activities are truly safe . the patients are unfamiliar with their capacity or the potential risks and restrictions ; therefore , it appears necessary to create professional teams dedicated to managing patients after chd repair . physical activity may be objectively assessed using a questionnaire including questions concerning the use of various exercise types of different intensity . for the purposes of this study , i.e. determination of the current physical activity profiles of patients , the data obtained from the stanford i and ii questionnaires were immensely useful in evaluating guch patients , even though the test was originally designed for other purposes [ 7 , 16 ] . the conducted analysis of the questionnaire 's results demonstrated that , after the completion of the rehabilitation program , the everyday physical activity of our patients increased . after the program ended , the subjects tended more often to report engaging in high - activity exercise ( the average stanford ii scores increased from 0.13 before the rehabilitation to 0.63 after the rehabilitation ) . it is , therefore , to be expected that , owing to this active attitude , the patients will be able to maintain the improvement of physical capacity and exercise tolerance achieved during the comprehensive cardiac rehabilitation program for a longer period of time . it should be underscored that the ultimate examination with the stanford questionnaires demonstrated that the patients undergoing comprehensive rehabilitation had statistically better results than the patients who refused to participate in the rehabilitation program ( mean scores : stanford i a : 3.2 vs. b : 1.8 ; stanford ii a : 1.2 vs. b : 0.13 , p < 0.001 ) . this may indicate that the elements of the program were properly selected and may confirm the program 's effectiveness , including in terms of the increase in awareness concerning the need for developing basic health - oriented habits and an active lifestyle . when analyzing the obtained responses , one should pay attention to the subjective opinions of patients concerning the benefits resulting from participating in the rehabilitation program . after completion of the training cycle , the patients declared that they felt much better , tired less , felt safer during physical activity , and did not fear engaging in types of activities that used to constitute an thus , it should be expected that , in the future , the group of patients who underwent the ccr - guch program will be more aware of their capabilities and will be better able to see their limits without overexerting themselves , which , unfortunately , tends to happen in the group of young adults with chds . the final results of the young adult guch patients were closer to the results of the controls , recruited ( as in previous studies conducted by the authors ) among young , healthy students . in accordance with the study 's methodology , cardiac rehabilitation conducted in the study group significantly improved the physical activity of over half of the participants . based on the obtained data , it can be surmised that the expected positive results of training and an improvement of the cardiovascular system 's adaptation to physical exercise were achieved during the program . the results confirm previously published literature data ; however , there are still few comprehensive studies devoted to the physical capacity of guch patients despite the growing number of such patients in the population [ 17 , 18 ] . the ccr - guch rehabilitation in the study group was designed in accordance with the guidelines for conducting physical training , published by the american heart association and with the experience of the authors in providing treatment and conducting physical training sessions for cardiovascular patients [ 3 , 14 , 22 ] . one limitation of the study was the lack of randomization during group selection ; however , the authors deemed refraining to invite the patients to participate in the program unethical . the number of patients included in the program was also restricted by the financial limits of the study . the intensity of physical exercise was determined based on the initial cpet . in order to maintain safety , the patients were monitored with electrocardiography during cycloergometer training , while the intensity of general fitness training and nordic walking sessions was controlled only by pulse measurements . in accordance with the premises of the ccr - guch program , acquiring self - control reduced anxiety related to continuing the activities and physical training in home conditions , without the supervision of physical therapists or physicians . in conclusion , it should be underscored that the improvement of the initially lowered physical activity of guch patients undergoing rehabilitation documented in this study , contrasted with the results of patients who did not undergo rehabilitation , confirms the need to qualify and include guch patients in comprehensive programs of late rehabilitation [ 19 , 20 ] . engaging in the comprehensive program of late cardiac rehabilitation improves the physical activity of patients after the surgical correction of congenital heart diseases . comprehensive cardiac rehabilitation reduces the fears concerning various forms of physical activity that are often encountered in this group of patients , and , in consequence , improves the patients quality of life . it appears justified to introduce the comprehensive rehabilitation program as a supplement to holistic care in the group of adolescent patients after congenital heart diseases .

introductionthe group of grown - up patients with congenital heart defects ( grown - up congenital heart guch ) complains of a number of specific medical and non - medical problems . the presented program of comprehensive cardiac rehabilitation ( ccr - guch ) , dedicated to the above mentioned group , can potentially improve the physical activity of guch patients.aimthe aim of the study was to assess the effect of the comprehensive cardiac rehabilitation program on the physical activity of guch patients.material and methodsthe invitation to take part in the ccr - guch program was addressed to a group of 57 patients ( mean age : 23.7 4.1 years ) who had undergone the surgical correction of ventricular septal defects ( vsd ) or atrial septal defects ( asd ) at least 12 months earlier . the patients were divided into two groups : a patients undergoing rehabilitation , and b patients who did not participate in the program . the patients were initially examined using functional and stress tests , and the program of comprehensive cardiac rehabilitation was started in group a. after 30 days , the patients from both groups underwent further testing using the same methods as during the initial evaluation.resultsafter one month of rehabilitation , the physical activity parameters of patients participating in the ccr - guch program ( group a ) were significantly better than those observed among non - participants ( group b).conclusionsthe introduction of the comprehensive rehabilitation program improves the physical activity and , consequently , the quality of life of guch patients . the ccr - guch program appears to be a justified supplement to holistic care in the late rehabilitation of patients after the surgical correction of congenital heart defects .

Wstp Cel Materia i metody Wyniki Wnioski Introduction Material and methods Comprehensive cardiac rehabilitation program Results Discussion Conclusions Disclosure

PMC4649102

traditional chinese medicines ( tcms ) , also known as botanical medicines or phytomedicines , have been used to promote health and treat diseases for over a millennium . differing from synthetic drugs , medicinal herbs and their preparations because of low toxicity and effective therapeutic performance , a rapid growth in worldwide demand for tcms has appeared in the last few decades , especially in asia , europe , and north america . as demand grows , the requirement for product quality is also improving . each batch of tcms should meet certain product specifications including both the time of production and over its shelf life . however , the quality control for tcms is more difficult than that for synthetic drugs , because even a simple preparation consisting of only a few herbs may comprise hundreds of mostly unique or species - specific compounds . complete characterization of all the compounds in a preparation is indeed a challenge , while selection of several compounds for determining either efficacy or quality is not in line with the principle of tcms . in recent years , significant efforts have been made to develop new analytic methods for quality control of tcms . among all quality control methods , chromatographic fingerprint , as a unique pattern indicating the information on multiple chemical components within a sample , could provide more informative and accurate assessment of complex matrix , such as tcm . in order to promote the valid fingerprint method for the quality control of herbal medicines , the china food and drug administration ( cfda ) suggested that all of herbal chromatograms should be evaluated by similarity , which is based on the calculation of correlative coefficient and/or cosine of vectorial angle ( i.e. , congruence coefficient ) between fingerprint data . now similarity evaluation is widely used as a close measure for assessment of chromatographic fingerprints in practice . however , similarity based on correlative coefficient or cosine of vectorial angle is the overall evaluation of fingerprints ; it is ambiguous and insensitive to the changes in content of components . that is , the similarity between chromatographic fingerprints could not sufficiently indicate the closeness in the contents of components , especially for low - content components . recently , hplc fingerprinting combined with quantitative determination of some characteristic compounds has been developed and validated for quality control of herbal preparations . although sample fingerprints shared a satisfactory similarity , the contents of some compounds between samples may still appear quite different ( i.e. , similar fingerprints do not mean similar components in content ) . this may indicate that the slight difference in similarity between fingerprints could suggest the large difference in amounts of components . it is unclear whether the difference in fingerprint similarity is significant or not . how to assess the difference in fingerprint similarity is worthy of investigating . to ensure the quality of tcms but for now it is unrealistic to identify and quantify all the chemical components in such a complex system as tcm . the xin - ke - shu ( xks ) tablet is one of the most commonly used tablets in tcms for treatment of coronary heart and cerebrovascular disease . it is a really complex matrix , which comprises five medicinal materials or extracts thereof , including salviae miltiorrhizae radix et rhizoma , pueraria lobatae radix , crataegi fructus , notoginseng radix et rhizoma , and aucklandiae radix . due to a high complexity in chemical composition , we developed a high performance liquid chromatography - diode array detector ( hplc - dad ) method for quantification of multiconstituent in xks tablet and found that the component contents in different batches of xks tablet had large differences . with the development of chemometric methods , a number of data processing tools were introduced and applied to process chromatographic fingerprints [ 1013 ] . in our present study , chromatographic fingerprint combined with chemometric methods was developed for batch consistency evaluation of xks tablet . firstly , four types of fingerprints characterized by different modes were used and compared by similarity analysis . according to the results of comparison , then , hierarchical cluster analysis was applied to observe whether class difference occurred in different batch samples . if high fingerprint similarity was achieved , but apparent class difference existed in samples , then mahalanobis distance analysis was employed to estimate the probability level of obviously different samples . subsequently , for the significantly different fingerprints of batches determined by mahalanobis distance , t - test was used to find the peaks with significant difference in areas . although these peaks found by t - test were based on peak areas , the results were theoretically identical to those based on concentration . the peaks having significant differences were identified by hplc / it - ms as well . finally , a useful strategy based on the combination of hplc fingerprint and chemometric methods was proposed for rational evaluation of batch - to - batch consistency of xks tablet and could be also useful for assessing the batch consistency of other tcms . hplc - grade acetonitrile and methanol ( meoh ) were purchased from merck company ( merck , darmstadt , germany ) . ultrapure water was purified with a milli - q system ( millipore , bedford , usa ) . ( tedia way , fairfield , usa ) . reagent grade meoh was purchased from tianjin kemiou chemical limited company ( tianjin , china ) . in order to include as many sources of sample variability as possible , seventy - one batches of xks tablet that were produced in a long time span of three years were provided by shandong wohua pharmaceutical limited company in china . all the xks tablet samples were kept in a cool room at 4c and 30% relative humidity with packages ; storage stability of xks tablet , both the physical and chemical properties , was validated . in the cool room , the chemical profiles and the concentration of the major compounds ( we determined 6 of the major compounds in xks tablet , details not shown here ) of xks tablet were consistent for 3 years . the pulverized powder of 100 mg for each sample was accurately weighed and ultrasonically extracted with 5 ml 70% methanol for 20 min in a conical flask at room temperature . this sample solution ( 4 ml ) was transferred into 10 ml centrifuge tube and centrifuged at 3000 rpm for 10 min . the supernatant was further filtered through a 0.45 m membrane filter before hplc injection . , santa clara , ca , usa ) with a diode array detector ( dad ) was used to acquire chromatograms and uv spectra . all of the chromatographic analysis was performed on a phenomenex c18 column ( 250 mm 4.6 mm i.d . 5 m ) protected by a precolumn ( 12.5 mm 4.6 mm i.d . 5 m ) of the same material . the mobile phase was composed of acetonitrile ( a ) and 0.1% formic acid in water ( b ) . the conditions of solvent gradient elution were 812% ( a ) in 021 min , 1217% ( a ) in 2131 min , 1738% ( a ) in 3155 min , 3890% ( a ) in 5565 min , and 90% ( a ) in 6575 min , at a flow rate of 1.0 mlmin . the uv detector was set at 278 nm with full spectral scanning from 190 nm to 400 nm at 2 nm step . the column temperature was maintained at 30c , and all the injection volumes of sample solutions were fixed at 10 l . santa clara , ca , usa ) was connected to hplc system via an esi interface . ultrapure helium ( he ) was used as the collision gas , and pure nitrogen ( n2 ) was employed as the nebulizing gas . the instrumentation and chromatographic condition of hplc for lc - ms were the same as those used for hplc - dad analysis . for identification of the interesting constituents in the fingerprints of xks tablet , the following conditions of ms analysis were used : negative ion mode ; capillary voltage 3.5 kv ; drying gas flow rate 12 lmin ; drying gas temperature 350c ; nebulizer 35 psi ; mass analyzer range was from m / z 50 to 1250 amu . all the data were processed using data analysis for 6300 series ion trap lc / ms ( version 3.4 ) . in order to investigate whether different types of fingerprints characterized by different modes have notable differences in characterization of xks tablet samples , four types of fingerprints were applied , including single wavelength fingerprint , 3d fingerprint , maximum absorption wavelength fingerprint , and multiwavelength fingerprint . the four types of fingerprints were developed as described in additional file 1 in supplementary material available online at http://dx.doi.org/10.1155/2015/589654 . hplc - dad data of the 71 batches of samples were obtained from agilent chemstation software . the software similarity evaluation system for chromatographic fingerprint of traditional chinese medicine ( version 2004a ) recommended by cfda ( the china food and drug administration ) was used to align the fingerprints of different batches and to simultaneously calculate similarity between samples . natick , ma , usa ) was used for handling chromatographic data matrices , calculating the mahalanobis distance and carrying out hierarchical clustering analysis ( hca ) principle component analysis ( pca ) and statistical test ( i.e. , t - test and f test ) . about similarity calculation , one is cosine of vector angle ( rcos ) that is often used ; the other is correlation coefficient ( r ) . both rcos and r were calculated for similarity evaluation of chromatographic fingerprints among different batches of samples . ward 's method was used for hca due to relatively powerful performance compared with other methods . mahalanobis distance is a distance measure in statistics and takes into account the correlated variables in data set , which can be written as(1)di2=xixs1xix,where xi , x- , and s are the ith sample , the mean vector ( i.e. , the centroid of class ) , and the variance - covariance matrix of a class , respectively . according to mahalanobis distance , the unknown samples can be classified as acceptable or unacceptable for the training set at a certain probability level [ 17 , 18 ] . critical values at a certain probability level ( ) are obtained by f distribution . the mahalanobis distance of a sample was compared with critical value to determine whether the difference of the sample relative to the mean of the training set was significant or not . the sample having a probability level in the range from 1.0 to 0.05 is classified as a member of training set . the sample having a probability level in the range from 0.05 to 0 is classified as an outlier . since the single wavelength fingerprint was often used in practice , the validation of hplc fingerprint method for xks tablet was performed on the basis of this type of fingerprint . the relative standard deviation ( rsd ) values of common peak areas and relative retention time of common peaks were in the range of 0.63.5% and 0.31.2% , respectively . the repeatability was estimated by running five independently prepared xks tablet samples , which were from the same batch , and the corresponding rsd values of common peak areas and relative retention time of common peaks were lower than 1.8% and 1.4% , respectively . a sample solution placed over 3 days in room temperature was employed to carry out the stability test . the corresponding rsd values of common peak areas and relative retention time of common peaks were less than 3.5% and 2.3% , respectively . the results of validation suggested that the developed hplc fingerprint method was satisfactory and applicable to sample analysis . the typical fingerprints ( single wavelength fingerprints ) of 71 batches of xks tablet samples after peak alignment were displayed in figure 1 . the similarity between the fingerprint of each sample and the standard fingerprint ( i.e. , the mean fingerprint of 71 batches of samples ) was shown in additional file 2 ( supplementary figures 1 and 2 ) . there are no apparent differences found in similarity trend along with the sample distribution for different types of fingerprints . although fingerprints are based on different characterization modes , the fingerprints of different batches of samples have a high similarity to the standard fingerprint ( all above 0.94 based on r or rcos ) . according to the literature , in addition to the maximum wavelength fingerprint , the other three types of fingerprints are considered to have good similarity for different batches of samples . the range and the degree of dispersion of similarities based on four types of fingerprints were summarized in table 1 . according to table 1 , the maximum wavelength fingerprint has the largest range and the dispersion degree of similarity as compared with the other three types of fingerprints . because this type of fingerprint is characterized with the data at maximum absorbance wavelength , the sensitivity is relatively high . the 3d fingerprint is very close to the single wavelength fingerprinting in the range and the dispersion degree of similarity . among four types of fingerprints , multiwavelength fingerprint exhibits the minimum range and dispersion degree of similarity due to the quite close distribution of multiactive ingredients in different batches of samples . up to now , the single wavelength fingerprint is the most used in practice . in this type of fingerprint , the particular wavelength the 3d fingerprint for characterizing each sample contained rich information on components ; however , redundant information was also present . additionally , poor resolution and unstable baseline appeared in some wavelengths , making the subsequent data processing ( such as integration and peak alignment ) time - consuming , difficult , and complicated . the multiple - wavelength fingerprint could have good reflection on characteristic components but lost some useful information on other constituents . the maximum wavelength fingerprint was a satisfactory type of fingerprint and could better reflect the differences in chemical composition between samples . however , it was not easy to get the maximum absorbance fingerprint , because some complicated and time - consuming data handling methods or special chemstations ( such as waters empower chemstation ) should be necessary . according to the comparison mentioned above , the single wavelength fingerprint seems to be still the most practical , popular , and easy - handling fingerprint pattern for quality evaluation of herbal medicine product . considering fingerprint applicability and representativeness , overall , the dendrogram revealed two clear clusters , suggesting that there were apparent class differences in different batches of xks tablet samples . although high similarity between samples was acquired ( supplementary figure 2 ) , the class differences seemed to be significant . in order to determine whether or not the differences between batches of xks tablet samples were statistically significant , mahalanobis distance method was employed . for calculation of mahalanobis distance good quality samples should be candidates of the reference class . in our study , a sample having larger peak areas in its fingerprint was selected as the member of the reference class due to large peak area generally indicating high content of component . sample 5 was first selected as a reference sample for development of the reference class , because most of peak areas in sample 5 fingerprint are larger as compared to other batches of samples . the euclidian distances between other batches of samples and sample 5 were then calculated for evaluation of sample closeness . the reference class was developed by choosing the closest 10 batches of samples plus sample 5 ; therefore , 11 samples were involved in the reference class according to the requirement of the documents on the development of fingerprint . the mean fingerprint of the samples in the reference class was also calculated as the reference fingerprint . with respect to the centroid of the reference class , the mahalanobis distance and its probability level of each sample in the reference class the results reveal that all sample fingerprints in the reference class have nonsignificant differences as compared to the reference fingerprint . the mahalanobis distances between the fingerprints of other batches of samples and the reference fingerprint were also calculated and shown in figure 3 . there are 19 batches of samples having significant differences with respect to the centroid of the reference class determined by mahalanobis distances , indicating that their fingerprints may have large differences in peak areas relative to the reference fingerprint . to explore the relationship between similarity ( both cosine of vector angle and correlation coefficient ) and mahalanobis distance , similarity , mahalanobis distance , and corresponding probability of each batch fingerprint relative to the reference fingerprint however , although the fingerprints of some samples ( such as samples 9 , 15 , and 20 , and their r and rcos are both above 0.99 ) have high similarity to the reference fingerprint , their corresponding mahalanobis distances are still larger , which result in significant differences ( < 0.05 ) . notice that higher similarity does not exactly indicate small mahalanobis distance , while lower similarity indicates larger difference in mahalanobis distance . according to ( 1 ) , the mahalanobis distance is actually based on the differences in peak areas between fingerprints . for r or rcos , although the chromatographic peak areas are used in similarity calculation , similarity indicates a measure of the closeness of the overall shapes of the compared fingerprint profiles . when the shapes of the two hplc fingerprints are very close , high similarity can be obtained even if the peak areas in the compared fingerprints are quite different ( e.g. , the peak areas vary greatly with different injection volumes in different fingerprints , but the similarity is still high owing to close shapes of fingerprint profiles ) . if the difference in peak area is very great and can significantly affect the shape of the compared fingerprints , the similarity between the fingerprints will change accordingly . therefore , the mahalanobis distance takes the difference of peak area into account , while similarity emphasizes the overall shape of the fingerprint profile . in some cases , although the fingerprints of some batches showed high similarity to the reference fingerprint , their differences in peak areas were still significant based on mahalanobis distances . therefore , the mahalanobis distance could reflect the inconsistency between batches of samples relatively better than similarity , and the corresponding probability level was also estimated for assessing whether the inconsistency between fingerprints was significant or not . if peak area has large difference in various batches of samples , quality consistency will be strongly affected . therefore , it is quite necessary to find the significant different peaks across the fingerprints of batches . the mahalanobis distance was used to detect whether there are significant differences between the sample fingerprints and the reference fingerprint . however , it is not easy to find the significantly different peaks in a sample fingerprint by mahalanobis distance . in our study , because common peaks are common features of different batches , differences in areas of common peaks will have great impact on quality consistency . moreover , for each sample fingerprint , the common peak areas also accounted for above 81% of the total peak areas . therefore , only common peaks were identified by t - test for significantly different batches determined by mahalanobis distance . more attention should be paid to the significantly different common peaks for the quality control of xks tablet . the reference class could be regarded as the training set . to carry out the t - test on the peaks in a sample fingerprint , the variable zi was calculated according to(2)zi = xixisi , where xi referred to the ith chromatographic peak area in a fingerprint of sample , and x - i and si were the average peak areas and the corresponding standard deviations of peak i in the fingerprints of the samples in the training set ( i.e. , the reference class ) , respectively . n was the number of samples in the training set ( n = 11 in the present study ) . the ti was compared with the tcrit given by student 's t distribution to determine whether the difference between xi and x - i was significant or not . although , t - test was calculated with chromatographic peak area , the result indirectly denoted the differences in component contents of different batches of samples , because , in the linear range , a chromatographic peak area was expressed by(4)xi = fici+bi , where xi and ci referred to the chromatographic peak area of peak i and its corresponding concentration ( or content ) , respectively . fi and bi denoted the response factor and the intercept of the calibration curve , individually . then , ( 2 ) combined with ( 4 ) could be written as(5)zixixisi = ficicifi1/n1i=1ncici21/2=cicisci , where c - i and sci represented the average content and the standard deviation of ith component concentrations in the samples of the reference class . so , according to ( 5 ) , the value of zi based on peak area was identical to that based on component content for peak i. figure 5 shows the z values of common peaks in 19 significantly different batches of samples determined by mahalanobis distance analysis . among the 19 batches of samples , most of common peak areas with significant differences identified by t - test are the small ones in peak areas , which indicate that the amounts of these components may be lower than the average contents of the corresponding components in the samples of the reference class . figure 6 shows the frequency proportion of significantly different common peaks based on t - test versus 19 batches of samples . the results clearly reveal the common peaks 13 and 19 having the highest frequency percentage that occurred in the 19 batches of samples . the components corresponding to the common peaks with high frequency percentage may have large differences in amount . hplc / it - ms was employed to identify seven of them ( peaks 13 , 15 , 16 , 19 , 20 , 26 , and 29 ) which displayed high frequency proportion in figure 6 and are marked in figure 1 . in the esi - ms experiment , the molecular weight of each peak and some fragments could be obtained . in negative ion esi mode , the peaks were detected according to the deprotonated molecules ( [ m - h ] and/or [ m - h + hcooh ] ) . except peak 26 , which was identified as lithospermic acid ( characterized based on the retention time , uv spectrum , and its ms data compared with literature ) , the other six interesting peaks were tentatively identified as isoflavonoids . the diagnostic ions of isoflavonoids are the loss of 162 da and 120 da in molecular weight , which are related to the o- and c - glycoside isoflavones [ 21 , 22 ] , respectively . the possible structures of peaks 13 , 15 , 16 , 19 , 20 , 26 , and 29 were characterized based on their retention times , uv spectra , and their ms data compared with literatures . using peak 15 as an example , the fragmentation of [ m - h ] ion at m / z of 445 gives product ions at m / z 325 [ m - h-120 ] , 297 [ m - h-120-co ] , and 282 [ m - h-120-co - ch3 ] , which indicate that peak 15 has a -och3 group in the aglycone skeleton and the neutral loss of 120 da characteristic of c - glycosides . more information about these seven peaks and their structures are shown in table 3 and figure 7 , respectively . according to ms results , lithospermic acid and six isoflavonoids as characteristic markers may have strong contribution to the inconsistency of batches of xks tablet samples . the isoflavonoids were the main active ingredients of pueraria lobatae radix , suggesting that the pueraria lobatae radix could have large fluctuations in the production of xks tablet . consequently , in order to improve the consistency of different batches of xks tablet , more attention should be paid to the quality of pueraria lobatae radix , including selection of good quality of raw materials , improvement of manufacturing technologies , and enhancement of process control level . to find a rational way to evaluate the consistency of different batches of xks tablet samples , the useful strategy was proposed for the solution of the routine issue existing in the consistency evaluation of chromatographic fingerprints of xks tablet samples . although high similarity occurred in some batches of samples , the significant differences between samples could still be found by mahalanobis distance . the chromatographic peaks with significant differences in peak areas determined by t - test could suggest that the corresponding constituents would have significant differences in content , which resulted in affecting the consistency of quality . our study demonstrated that the proposed strategy based on the combination of hplc fingerprints and chemometric methods would provide a useful way to assess the quality consistency of xks tablet .

evaluation of the batch consistency of traditional chinese medicines ( tcms ) is essential for the promotion of the development and quality control of tcms . the aim of the present work was to develop a useful strategy via liquid chromatography and chemometrics to evaluate the batch consistency of tcm preparations . xin - ke - shu ( xks ) tablet was chosen as a model for this method development . four types of chromatographic fingerprint approaches were compared by using similarity analysis based on cosine of angel or correlation coefficient . differences in the fingerprints of 71 batches of xks tablet were illustrated by hierarchical cluster analysis . then , mahalanobis distance was employed for estimating the probability level ( p < 0.05 ) of the differences mentioned above . additionally , t - test was applied to find out the chromatographic peaks which had significant differences . for xks tablet , the maximum wavelength fingerprint had the largest range and dispersion degree of similarity as compared with the other three ones . there were two clear clusters in all the batches of samples . and we clearly arrived at the conclusion that higher similarity does not exactly indicate small mahalanobis distance , while lower similarity indicated larger mahalanobis distance . finally , a useful strategy was proposed for evaluation of the batch consistency of xks tablet .

1. Introduction 2. Methods 3. Results 4. Conclusion

PMC5287170

lentinula edodes was cultured in 500 ml of sterile potato dextrose broth at 25 for 28 days with standing . as shown in fig . 1 , the culture filtrate exhibited antibacterial activity , forming a clear zone of 22 mm on the paper disk method against ralstonia solanacearum , a causal agent of tomato bacterial wilt disease . to purify the antibacterial compound , the culture filtrate was subjected to octadecyl - silica column chromatography and eluted with different concentrations from 5% to 100% methanol . none of the 50-l aliquots of methanol eluate showed an antimicrobial effect on the bacteria , but the water fraction that passed through the column maintained antibacterial activity . the ph of the passed fraction was 3.9 , suggesting the presence of an organic acid . therefore , qualitative analyses of organic acids from the culture filtrate of l. edodes were carried out by high - performance liquid chromatography ( hplc ) . hplc was performed on a hitachi chromaster apparatus ( tokyo , japan ) , which consisted of a pump , autosampler , column oven , uv - vis detector , and hpx-87 column ( i.d . 4.6 300 mm , particle size of 5 m , aminex therapeutics , kenmore , wa , usa ) ; temperature was maintained at 25. the flow rate was 0.6 ml / min . the mobile phase was 4 mm h2so4 in water for a total running time of 25 min . qualitative analysis of organic acids in the culture filtrate of l. edodes was performed by hplc and the results were compared with those of authentic organic acids . oxalic acid was a dominant component in the culture filtrate of l. edodes , accounting for 50% of the hplc analysis data . in addition to oxalic acid , phytic acid , malonic acid , and fumaric acid were major components . quantitative analysis of oxalic acid in the culture filtrate of l. edodes was carried out under the same hplc conditions as were used in qualitative analyses . for quantitative analysis , a standard curve of oxalic acid was prepared in the concentration range of 200~2,000 g / ml using four different concentrations . the coefficient of the correlation value for the standard curve of oxalic acid was 0.9982 . thus , the content of oxalic acid in the culture filtrate of l. edodes was 0.07% . louis , mo , usa ) was tested for its antibacterial activity against r. solanacearum using the paper disk method . 5,000mg / l ) were added to the filter paper disks ( 5 mm diameter ) , placed on nutrient agar , and overlaid with soft agar ( 0.8% ) mixed with bacterial cells ( 5 10 ) . clear zones that had formed around the paper disks were evaluated as growth inhibition of the bacterial cells . the antibacterial activities of nine different organic acids present in the culture filtrate of l. edodes shown in fig . 2 were examined against r. solanacearum . each clear zone of 34 , 27 , and 12 mm was formed on concentrations of 5,000 , 100 , and 500 mg / l , but not on other concentrations of oxalic acid . antibacterial activity on 500mg / l oxalic acid was compared to the positive control , tetracycline ( 30mg / l ) . furthermore , the antibacterial effect of oxalic acid was evaluated against eight different phytopathogenic bacteria : xanthomonas campestris pv . vesicatoria , escherichia coli , and a gram - positive bacteria strain , bacillus subtilis . different concentrations of oxalic acid from 300 to 100mg / l were added to nutrient broth containing different bacterial cells and cultured at 28 for 24 hr . as shown in table 1 , oxalic acid exhibited antibacterial activities against the bacterial strains tested at the minimum inhibitory concentration ( mic ) of 250mg / l , except x. campestris pv . vesicatoria were the most sensitive , and cell growth was inhibited at an mic of 200mg / l . interestingly , oxalic acid , like antibiotics such as tetracycline , had a bactericidal effect and the growth of bacterial cells could not be recovered on nutrient media . in preliminary experiments , the ph of media containing oxalic acid was ph 3.5~4.5 ; we further evaluated whether the antibacterial activity of oxalic acid was a result of low ph . the ph 3.5~4.5 media were adjusted with hcl and the r. solanacearum cells were cultured in the media . the bacterium was grown in the media , although the growth rate was slightly different corresponding to ph ( data not shown ) , indicating that low ph is not main factor affecting antibacterial activation . taken together , the results suggest that oxalic acid is critical factor responsible for antibacterial activity . oxalic acid , a saturated short chain dicarboxylic acid , occurs naturally in many plants and fungi and forms insoluble calcium or magnesium oxalate crystals , soluble sodium , or potassium oxalate . in previous study , different organic acids were identified in japanese apricot fruit by hplc analysis and their antibacterial activities were determined against e. coli , b. subtilis , staphylococcus aureus , and streptococcus suis . however , oxalic acid showed relatively low antibacterial activities towards these bacterial species with an mic of 1,300mg / l compared to the mic of 500 mg / l found in this study . oxalic acid produced by plant pathogenic fungi provide a low ph , which results in maximal activities of fungal enzymes that degrade the cell wall of the host plant . in artificial cultivation of l. edodes mushroom , l. edodes may degrade wood sawdust by producing lignocellulytic enzymes ( laccase , cellulase , and xylanase ) and the resulting product , glucose , is used in energy metabolism . the sms after 4 cycles of harvest was obtained from a mushroom farm , mixed with water ( 1 : 3 ratio w / v ) , and incubated with shaking for 2 hr . the mixture was then filtered through two layers of miracloth ( calbiochem , la jolla , ca , usa ) , centrifuged for 10 min at 5,000 g and the supernatant was used as the water extract of sms . water extract from the natural mushroom substrate that had not been inoculated with l. edodes was used as a control . to quantitatively analyze oxalic acid , the one peak at 6.63 min indicating oxalic acid was detected at 1000 mau in the water extract of a sms sample , but not in the water extract from natural mushroom substrate . thus , the extract of sms of l. edodes may be an eco - friendly control agent for bacterial plant diseases .

the culture filtrate of lentinula edodes shows potent antimicrobial activity against the plant pathogenic bacteria ralstonia solanacearum . bioassay - guided fractionation was conducted using diaion hp-20 column chromatography , and the insoluble active compound was not adsorbed on the resin . further fractionation by high - performance liquid chromatography ( hplc ) suggested that the active compounds were organic acids . nine organic acids were detected in the culture filtrate of l. edodes ; oxalic acid was the major component and exhibited antibacterial activity against nine different phytopathogenic bacteria . quantitative analysis by hplc revealed that the content of oxalic acid was higher in the water extract from spent mushroom substrate than in liquid culture . this suggests that the water extract of spent l. edodes substrate is an eco - friendly control agent for plant diseases .

Identification of antibacterial substances Quantitative analysis of oxalic acid Antibacterial activity of oxalic acid

PMC3612492

clinically , frontal lobe syndromes , frontal network syndromes , frontal systems syndromes , executive dysfunction , and metacognition have all been used to describe disorders of frontal lobes and their extended networks although they are not all synonymous . however , because the frontal lobes network with every other part of the brain , strictly speaking , frontal network syndromes constitute the most accurate neurobiological depiction . the term , frontal network syndromes ( fns , ) emphasizes the universal connectivity of the frontal lobes with all other brain regions . for example , the stroke literature is replete with fns that have been reported with discreet lesions outside the anatomical boundary of the frontal lobe , such as subcortical grey matter , subcortical white matter , with isolated lesions of the brainstem , cerebellum , temporal , and parietal lobes [ 18 ] . for the purposes of simplification , five primary syndromes and numerous secondary syndromes may be delineated . impairment in working memory , executive function , abulia , disinhibition , and emotional dyscontrol may be regarded as the elementary deficits of fns . in addition , a number of secondary manifestations may be identified such as a wide array of behavioral abnormalities such as loss of social norms , imitation behavior , compulsions , and obsessions [ 9 , 10 ] ( figure 1 ) . to begin to understand the most complex object in the universe , the human brain and in particular the frontal lobes , it is most illuminating to study the evolution of our mind and thereby gain a better understanding of the clinical syndromes we are faced with today . in the words of theodosius dobzhansky , nothing in biology makes sense except in the light of evolution . life on earth evolved approximately 3.7 billion years ago and thereafter continuously shaped by extra - terrestrial and geological events , punctuated by a number of key events . the inclusion of prokaryotes into eukaryotic cells furnished cells with a powerhouse , the mitochondria . some time after snowball earth , when glaciers reached the equatorial regions about 620590 million years ago ( mya ) , with the cambrian explosion of organism diversity , vertebrates ( bony fish , amphibians , reptiles , birds , and mammals ) formed ( ~520 mya ) . formation of the vertebrate skeleton allowed rapid movement , an advanced nervous system , and high degree of encephalization even though 98% of animal species are invertebrates versus 2% being vertebrate . myelination enabled a vastly improved neural transmission , speeding up neural transmission by a factor of 10 ( ~9 meters per second in unmyelinated fiber versus 50100 meters per second in myelinated fiber ) , with increased temporal precision , faster communication between the brain and body parts , and ability to react more rapidly to prey and predator [ 13 , 14 ] . with warming conditions , fish evolved lungs and walked on land about 365 mya , mammalian evolution ( ~200 mya ) and subsequent proliferation after dinosaur extinction ( ~65 mya ) . east side story event ( african rift valley formation leading to a hot and dry east africa ) precipitated bipedalism , increase in brain size , and tool making . the emergence of dopamine as a key neurotransmitter was critical in cooling our bodies and brains in a thermally stressed environment and later exapted for executive function . around this time , our frugivorous diet ( since ~60 mya ) was supplemented with meat and with the advent of marine isotope stage 6 ( 180120 mya ) that may have also served as an important event that highlighted the key dietary changes to sea - food that may have played a factor in advancing our cerebral connectivity that ultimately made us modern humans [ 16 , 17 ] . shell fish ( scallops oysters and prawns ) are rich in both iodine and essential fatty acids , both of which have been correlated with boosting dopamine activity and intellectual development . as a starting point using the so - called missing link hominid , australopithecus africanus ( brain volume approximately 450 ml ) , there was an increase in size to approximately 1500 ml in neanderthals over a 3 million year period and subsequently a slight decrease again in modern humans homo sapiens sapiens to 1350 ml . during this time , there was a reduction in the size of the visual striate area ( ba 17 ) with a relative increase in the posterior parietal cortices and frontal lobe reorganization at the network , neurotransmitter , and receptor levels . the size of frontal lobes in various mammalian species is frequently cited as steadily progressing allometrically from the so - called lower forms ( rats and mice ) to dogs and cats with primates and humans having the biggest proportionally . the frontal lobes comprise of 37%39% of the cerebral cortex macroscopically and connect to all other parts of the brain , often in a reciprocal manner . the frontal lobe in humans is as large as that would be calculated for an ape of human brain size overall , not larger as is often reported . however , what sets us apart from other mammals is not so much brain size but reorganization of our brains in terms of connectivity and neurotransmitter changes . these changes may be summarized in the following manner.progressive increase in size.hemispheric asymmetry , also called cerebral torque ( right frontal and left occipital petalias).neuropil reorganization.reorganization in terms of neurotransmitter systems.receptor modification . hemispheric asymmetry , also called cerebral torque ( right frontal and left occipital petalias ) . axons , dendrites , and space between the neurons and glial cells constitute the neuropil which is decreased to ba 10 in humans relative to other primates . in broca 's area ( ba 44 , 45 ) , the cortical architectural units or minicolumns are wider in humans relative to primates . ba 10 is twice as large in terms of overall brain volume compared to any of the other great apes ( 1.2% in humans versus 0.46%0.74% in great apes ) . spindle cells or von economo cells appear in layer vb in both the fronto - insular cortex as well as the anterior cingulate cortex , only in humans and african ( not asian ) great apes and are approximately 30% more numerous in the right hemisphere of these species . as they arose in our last common ancestor about 10 mya , they probably subserve the role of social and emotional processing which arose many millions of years before language . in view of this as an example of convergent evolution , other intelligent species such as the cetaceans have spindle cells . the neuronal density of the important ba 10 and 13 in humans is about half ( human ba 10~32000 and ba 13~30000 neurons per cubic millimeters ) that of the great apes ( chimpanzee ba 10~60000 and ba 13~43000 ) and is often much less than half ( orangutan ba 10~78000 neurons per cubic millimeter ) . the increase in neuropil particularly of ba 10 is likely related to the connectivity of this region with other tertiary association cortex and the other hemisphere . surprisingly , this cortical region is larger in size in humans than would be predicted for an ape of human brain size . there is a relative increase in the size of white matter , and the ratio of gyral - to - core white matter in the temporal lobes is larger than would be predicated for other hominoids . this relatively enlarged gyral white matter compared to core white matter is interpreted as reflecting greatly increased interconnectivity subserved by short association fibers . of the component amygdaloid nuclei ( lateral , basal , and accessory nuclei ) , the lateral nucleus is relatively larger in humans than would be expected in an ape of human brain size . this has been attributed to the increased interconnectivity with the temporal lobe 's unimodal and polymodal sensory information . in human evolution , there has been a differential expansion and reorganization not only of the temporal lobes and amygdaloid complex , but also of the inferior parietal lobes . this implies that evolution may have acted on neural systems rather than discrete anatomical structures . areas that are critical to social behavior which include the amygdaloid nuclei and limbic component of the frontal cortex are both volumetrically larger and revealed reorganization in their networks . this is in direct contrast to the traditional view that limbic structures are conserved whereas the frontal lobes had enlarged . within the frontal lobes themselves , however , many organizational and network changes have of course taken place . structures and networks that are implicated in social and emotional processing include the orbitofrontal cortex , the amygdala , fronto - insular cortex , and temporal polar cortex with the latter also important in language processing . these represent the so - called mosaic reorganization that has been a feature of human evolution . the quick acting excitatory ( glutamate ) and inhibitory ( gaba ) neurotransmitters ( nt ) act via ion channels with charged ions enabling a relatively quick response in terms of microseconds and seconds . the neuromodulators ( nm ) such as serotonin ( 5-ht ) , dopamine ( da ) , norepinephrine ( ne ) , acetylcholine ( ach ) , and histamine ( h ) act differently in that they promulgate longer lasting and more diffuse actions via the g - protein cascade system . the ultimate outcome on a network may be excitatory , inhibitory activation of their own presynaptic autoreceptors or interaction synergistically with the other nm systems . different nt 's subserve different higher cortical functions ( hcf ) , and in neurodegenerative disease nt deficits occur in varying combinations . the downside of nt modulatory systems for intellectual advances probably made humans more susceptible to a number of neurodegenerative diseases , unique to humans . within the frontal subcortical circuits , the reasons for this have been proposed as part of a very plausible and well - researched hypothesis by fred previc and their open loop systems ; da and ach became the predominant nt 's in the left hemisphere and ne and 5ht in the right hemisphere . da is considered to have been one of the key factors in the emergence of human intelligence . east side story with east african becoming relatively dry and arid , heat management and combating the deleterious effects of hyperthermia ( including so called heat stroke ) were a critical factor in survival of the mammals and the newly emerged bipedalist , australopithecus africanus . the function of da in lowering body temperature presumably enabled early hominoids to better tolerate the hyperthermia of chase hunting and catching prey that succumbed to chase myopathy [ 52 , 53 ] . thereafter in an evolutionary sense , da expansion occurred , due to increased calcium metabolism from prolonged aerobic activity as well as the increased tyrosine ( a dopamine precursor ) consequent to increasing meat supplementation about 2 mya . the clinical sequelae of blocking dopamine ( by drugs such as haloperidol , risperidone , and quetiapine ) as malignant hyperthermia and neuroleptic malignant syndromes may be therefore interpreted in an evolutionary perspective . da became exapted as the most important nt in our evolving brains , eventually concerned with most of the core frontal functions working memory , cognitive flexibility , motor planning , abstract representation , temporal sequencing , and generativity . da exerts a modulatory effect ( affects signal - to - noise ratio ) on the pfc g - protein linked receptors on dendritic shafts and spines of glutaminergic pyramidal neurons and dendrites of gaba - ergic neurons . humans and great apes feature da input to all cortical areas , in contradistinction to the paucity of da - ergic innervation of rodents . this was determined by measuring cortical da innervation ( axon density ) using tyrosine hydroxylase immune reactivity . in addition , there is a regional da - ergic distribution most intense in layer i and v - vi of the association cortices . furthermore , compared to great apes , humans have a generalized increased da - ergic input to the prefrontal cortical regions . the dopaminergic hypothesis in human evolution purports that the expansion of human da - ergic in particular was the most important factor in human tool making , exploration , cultural , and scientific developments . this theory also proposes that the drawbacks are the propensity for hyperdopaminergic syndromes such as schizophrenia , bipolar disease , autism , attention deficit hyperactivity disorders , and neurodegenerative diseases . in humans and great apes , compared to other mammals , there is an overall increase in the cortical output 5-ht efferents . the 14 different serotonergic g - protein - related receptors and one ion channel receptor ( 5-ht 3 ) enable the modulation of several different functions simultaneously , including memory , learning , and inhibition . this occurs via receptors on pyramidal cells and dendritic shafts and via interneurons which allows signal modulation from local circuits with reference to extrinsic stimuli . from a clinical point of view , serotonin in the ofc circuitry has been linked to self - control , emotional , processing , and inhibition regulation . in the neuromodulatory axons of humans and great apes exist varicose type axons that are likely to have a role cortical plasticity , associated with advanced traits such as superior learning capability , social learning , advanced tool manufacture , and self - awareness . these effects are mediated via 5 muscarinic receptors ( m1m5 ) , all of which are g - protein linked . nicotinic receptors are all ligand gated ion channels . both transmit mediating excitatory and inhibitory effects on gaba interneurons and pyramidal cells . hominoids are able to imitate behavior , and imitation ability of primates and hominoids was crucial to the cultural evolution . the imitation ability may be termed an all purpose learning mechanism . mosaic in that it involves the pfc , parietal , temporal , and cerebellar regions . the core features of memory and attention as well as more specific cognitive domains such as language and tool use are presumed to be based on the mosaic pattern , itself based on the imitation behavior circuitry that may have a visual , auditory , and tactile dimension . the fast acting excitatory ( glutaminergic ) and inhibitory ( gaba ) amino acid neurotransmitters are modulated by a number of widely projecting slower acting ( most g - protein linked ) neurotransmitters . this type of chemical architecture is useful in coordinating many neurons and neuronal circuits in response to a stimulus or threat . there are currently 8 chemical or neuromodulatory tracts ( da , 5-ht , ne , ach , h , oxytocin / vasopressin , and orexin ) ( figure 3 ) with their nuclei of origins in the brainstem , basal forebrain or hypothalamus , and extensive cortical ramifications , two examples of which are depicted in figures 3 and 4 [ 1 , 2 ] . the main functions of the frontal lobes are motor action and the temporal integration of behavior . frontal lobe evolution may be seen as progressive refinement of pyramidal pathway responses ( motor , speech , and behavior ) by incorporating cognitive and emotional processes . optimal decision - making requires a flexible system that can incorporate a wide range sensory inputs , at the same time prioritizing and choosing the most effective response in a changing environment . this is achieved through the major cerebral network systems : superior frontal occipital fasciculus , superior longitudinal fasciculus , inferior longitudinal fasciculus , cingulum and uncinate fasciculus , and seven frontal subcortical circuits [ 63 , 64 ] . the major fasciculi are depicted in figure 5 and can also be imaged by diffusion tensor imaging with directional specificity that is color - coded.u fibers ( orange).superior occipito - frontal fasciculus ( royal blue).superior longitudinal fasciculus ( pink).inferior longitudinal fasciculus ( dark green).perpendicular fasciculus ( dark blue).uncinate fasciculus ( purple).arcuate fasciculus ( light blue)note two tracts.corpus callosum ( brown).cingulum ( red).fornix ( light green ) . namely , cortex - caudate - globus pallidus - thalamus cortex ( figures 6 and 7 ) . confusion of terminology may exist because of the rather cumbersome terminology related to subcortical structures , best delineated by a diagrammatic representation ( figure 8) . the neurotransmitters and neuropeptides integrated in this circuitry are mainly excitatory ( glutamate ) and inhibitory ( gaba ) being the principal ones but many others involved in a neuromodulatory capacity including the monoamines , enkephalin , neurotensin , substance - p , dynorphin , adenosine , and neuropeptide - y [ 9 , 6567 ] . direct and indirect pathways.connections with the other circuits ( corticocortical).connections to areas outside the fsc 's . direct and indirect pathways . glutamate release occurs from frontal cortical regions to the striatum , mostly caudate nucleus , less often to putamen and nucleus accumbens . this releases gaba at internal segment of gp and sn ; gaba from gpi ( globus pallidus interna ) to thalamus diminishes and in turn the thalamus increases glutaminergic excitation of cortical regions . the striatal neurons within this pathway that project to the gpi are termed striosomes with d1 receptors . the net result is a thalamic disinhibition [ 9 , 65 , 66].the indirect pathway balances the direct pathway . here the striatal efferents are termed matrix efferents wirt d2 receptors and project from the globus pallidus externa ( as opposed to interna ) with a net thalamic inhibition . some of the gpe neurons are cholinergic but most are gaba - ergic with two different types of gaba ergic cell types within the gpe termed gp - ti and gp - ta , which mediate crosstalk between the afferent and efferent circuits of the gpe as well as the direct and indirect pathways .connections to regions outside the fsc 's include ( i ) dlpfc ( ba 46 ) to parietal ( ba 7 ) , ( ii ) anterior cingulate to temporal lobe ( hippocampus , amygdala , entorhinal cortex ) , ( iii ) medial ofc to temporal lobe and iv ) lateral ofc to heteromodal sensory cortex [ 9 , 69 ] direct pathway . glutamate release occurs from frontal cortical regions to the striatum , mostly caudate nucleus , less often to putamen and nucleus accumbens . this releases gaba at internal segment of gp and sn ; gaba from gpi ( globus pallidus interna ) to thalamus diminishes and in turn the thalamus increases glutaminergic excitation of cortical regions . the striatal neurons within this pathway that project to the gpi are termed striosomes with d1 receptors . the striatal efferents are termed matrix efferents wirt d2 receptors and project from the globus pallidus externa ( as opposed to interna ) with a net thalamic inhibition . some of the gpe neurons are cholinergic but most are gaba - ergic with two different types of gaba ergic cell types within the gpe termed gp - ti and gp - ta , which mediate crosstalk between the afferent and efferent circuits of the gpe as well as the direct and indirect pathways . connections to regions outside the fsc 's include ( i ) dlpfc ( ba 46 ) to parietal ( ba 7 ) , ( ii ) anterior cingulate to temporal lobe ( hippocampus , amygdala , entorhinal cortex ) , ( iii ) medial ofc to temporal lobe and iv ) lateral ofc to heteromodal sensory cortex [ 9 , 69 ] there is a complex interplay of the neurotransmitters , receptors , and circuit plasticity such as enhancement of striatal dopamine release by cholinergic agonists . the importance of recognizing and understanding this circuitry is in the relationship they have to clinical syndromes clinicians appreciate with regard to frontal pathology . these may be conveniently conceived of as three principal syndromes of the following.predominant dorsolateral prefrontal cortex ( dlpfc ) : temporal organization of information , executive function , working memory , and multitasking . impairments here lead to akinetic mutism and the abulic spectrum of disorders .orbitofrontal ( ofc ) circuitry : the cortical component of the limbic system where the emotional and other limbic components are integrated into behavioral output . hence , disinhibitory syndromes may occur in response to lesions of the medial as well as the lateral ofc . personality change in the context of frequently normal cognitive ( dlpfc ) function is usual and a manifold of presentations is encountered principally . the various forms of echopraxia or imitation behavior , utilization behavior , and environmental dependency syndromes ( field - dependent behaviors ) are sometimes the overriding clinical manifestations of these lesions . from a neurophysiological point of view , the medial ofc attaches emotional valence to events in turn determines the strength of the episodic memory . impaired autonomic and endocrine processing is associated with lesions of the medial ofc .lateral ofc lesions have been correlated with ocd , depression , irritability , mood disorders , and field - dependent behaviors . obsessive compulsive disorders spectrum group of disorders include ocd spectrum itself that comprises of obsessions ( intrusive urges thoughts , images ) , compulsions ( repetitive , ritualistic type of activities of a physical or cognitive nature ) , tourette 's syndrome , kleptomania , risk seeking behavior , pathological gambling , body dysmorphic disorder , and the immune disorder of pediatric autoimmune neuropsychiatric disorders ( pandas ) [ 73 , 74 ] . predominant dorsolateral prefrontal cortex ( dlpfc ) : temporal organization of information , executive function , working memory , and multitasking . orbitofrontal ( ofc ) circuitry : the cortical component of the limbic system where the emotional and other limbic components are integrated into behavioral output . hence , disinhibitory syndromes may occur in response to lesions of the medial as well as the lateral ofc . personality change in the context of frequently normal cognitive ( dlpfc ) function is usual and a manifold of presentations is encountered principally . the various forms of echopraxia or imitation behavior , utilization behavior , and environmental dependency syndromes ( field - dependent behaviors ) are sometimes the overriding clinical manifestations of these lesions . from a neurophysiological point of view , the medial ofc attaches emotional valence to events in turn determines the strength of the episodic memory . impaired autonomic and endocrine processing is associated with lesions of the medial ofc .lateral ofc lesions have been correlated with ocd , depression , irritability , mood disorders , and field - dependent behaviors . obsessive compulsive disorders spectrum group of disorders include ocd spectrum itself that comprises of obsessions ( intrusive urges thoughts , images ) , compulsions ( repetitive , ritualistic type of activities of a physical or cognitive nature ) , tourette 's syndrome , kleptomania , risk seeking behavior , pathological gambling , body dysmorphic disorder , and the immune disorder of pediatric autoimmune neuropsychiatric disorders ( pandas ) [ 73 , 74 ] . personality change in the context of frequently normal cognitive ( dlpfc ) function is usual and a manifold of presentations is encountered principally . the various forms of echopraxia or imitation behavior , utilization behavior , and environmental dependency syndromes ( field - dependent behaviors ) are sometimes the overriding clinical manifestations of these lesions . from a neurophysiological point of view , the medial ofc attaches emotional valence to events in turn determines the strength of the episodic memory . lateral ofc lesions have been correlated with ocd , depression , irritability , mood disorders , and field - dependent behaviors . obsessive compulsive disorders spectrum group of disorders include ocd spectrum itself that comprises of obsessions ( intrusive urges thoughts , images ) , compulsions ( repetitive , ritualistic type of activities of a physical or cognitive nature ) , tourette 's syndrome , kleptomania , risk seeking behavior , pathological gambling , body dysmorphic disorder , and the immune disorder of pediatric autoimmune neuropsychiatric disorders ( pandas ) [ 73 , 74 ] . in addition to these three principal behavioral fsc 's , two others have recently been added . the inferotemporal subcortical circuit may be associated with deficits in visual discrimination , visual scanning , visual hallucinations , and psychosis . in addition , a circuit between the posterior parietal region ( ba 7 ) and the prefrontal region ( ba 46 ) also contributes to processing of visual stimuli of significance and accordingly visuospatial processing . individual lesions or disease processes usually affect more than one to differing degrees and in various combinations . to facilitate the diagnostic components and sometimes treatment options , it is useful to consider the fsc 's and their clinical counterparts in terms of neurophysiological core components and in correlation to the clinical syndromes ( figure 2 ) . from a clinical point of view , a vast panoply of symptoms have been associated with frontal lobe lesions but may conveniently grouped under the following primary domains . these include initiation , disinhibition , working memory , attention , monitoring , language , and emotional control . in addition , loss of creativity , curiosity , and initiative may also be present disinhibition . these include ( ub , ib , eds ) loss of judgment , loss of insight , impairment comportment , inappropriate social behavior , loss of empathy , irritability , aggression , irascible , excessive jocularity , irresponsible behavior , restlessness , hyperactivity , hypersexuality , hyperorality , and incontinence . this include verbal and nonverbal working memory , multitasking , abstract thought , planning ahead , temporal sequence of events . examples of executive functions include learning new information both verbal and visuospatial , searching memory systems , activation of past memories , temporal organization of behavior , attention , and generation of motor activity that includes speech , writing , or limb movement . these involve at least four of the five subprocesses : task setting , initiation of the task , monitoring , error detection , and behavioral self - regulation . these are , therefore , modulated by up to four of the fsc 's with task setting correlating with left dlpfc and monitoring / error detection correlating with right dlpfc activity . initiation of a new task involves both left and right superior medial frontal fsc 's and behavioral regulation disrupted after medial ofc circuit lesions . cerebral lesions distinct from the fsc 's may be associated with fns such as parietal lesions . for example , caudate lesion patients may have poor recall but relative preservation of recognition . thalamic lesion patients on the other hand may have impairment of recall and recognition . attention . under this rubric regarding differentiating attention and working memory ; attention allows certain stimuli be they sensory or cognitive to be given preference over other competing ones . working memory refers to the keeping a limited amount of information usually for a few seconds to allow manipulation or use of that information for another task . selective attention maintain behavioral or cognitive set in context of distracting or competing stimuli . alternating attention mental flexibility that allows individuals to shift their focus of attention and move between tasks . highest level of attention with ability to respond simultaneously to multiple tasks [ 78 , 79 ] . focused attention respond discretely to specific visual , auditory tactile stimuli . selective attention maintain behavioral or cognitive set in context of distracting or competing stimuli . alternating attention mental flexibility that allows individuals to shift their focus of attention and move between tasks . divided attention highest level of attention with ability to respond simultaneously to multiple tasks [ 78 , 79 ] . monitoring . this includes , broca 's aphasia ( lh ) , expressive aprosodia ( rh ) , transcortical motor aphasia , aphemia , and central aphasia . clinical lesions studies have implicated in particular the orbitofrontal cortex as part of the neural network for emotional responses . patients with orbitoprefrontal and medial frontal regions were significantly impaired in both cognitive and affective empathy as compared to parietal patients and healthy controls and those with damage restricted to the prefrontal cortex , no matter which side , resulted in impaired empathy and lesions involving the right parietal lesions . subsequently from a registry analysis a much more widely distributed lesion site network impairs ei , in keeping with the extensive contemporarily appreciated neurobiological emotional network proposed by pessoa ( table 6 ) . many different brain lesions may affect ei , including frontal , temporal , subcortical , and even subtentorial stroke syndromes with the strongest relationship ( ei scores ) pertained to the frontal and temporal regions ( table 2 ) . however , they are all frontal network syndromes that just happen to be treated by differing brain - related clinicians for historical reasons . frontal network syndrome phenotypes may comprise of a mixture of the primary and secondary syndromes in various combinations . clinical syndromes are also treated by different disciplines , with considerable overlap by psychiatry , neurology , neuropsychology , speech and language and physical medicine , and rehabilitation . it may be readily appreciated that the construction of many clinical scales we use such as the fab scale , the new consensus criteria for the behavioral variant of fronto - temporal lobe degeneration , and frontal network testing format of the coconuts are all derivatives of these core and secondary syndromes . there are other frontal network presentations that are not readily classifiable under the above system . as a brain lesion may cause both hypo- or hyperfunction of a circuit or a lesion may cause hypo- or hyperfunction remotely ( diaschisis ) because of the hodological nature of brain function or connectomics . one theory termed the paradoxical functional facilitation proposes that one brain area reverses inhibition in other areas or results in compensatory augmentation , resulting in counter intuitive paradoxical improvement in certain functions function . according to this theory , examples of such syndromes include the following.(i ) emergent artistic ability in the setting of neurodegenerative disease has been reported in association with frontotemporal lobe disorders , stroke , alzheimer 's disease , parkinson 's , epilepsy , and migraine .(ii ) delusional misidentification syndromes , seen particularly with right frontal stroke .(iii ) increased humor , particularly after right frontal lesion such as stroke .(iv ) loss of visual imagery in dreaming .(v ) savant syndromes that may include the sudden , acquired prodigious , sudden , splinter , or talented subtypes . emergent artistic ability in the setting of neurodegenerative disease has been reported in association with frontotemporal lobe disorders , stroke , alzheimer 's disease , parkinson 's , epilepsy , and migraine . delusional misidentification syndromes , seen particularly with right frontal stroke . increased humor , particularly after right frontal lesion such as stroke . savant syndromes that may include the sudden , acquired prodigious , sudden , splinter , or talented subtypes . the time pressured nature of clinical practice , limited interview time , emergency room evaluation , and patient cooperation all place constraints on the nature of tests and how much testing can be performed . hence it is useful to consider available tests in a time - orientated hierarchical manner . the overall decision on how to deploy more time consuming neuropsychological tests is detailed in the recommendations by the aan neuropsychological testing guidelines . an emerging viewpoint is that subcortical processes , mostly cerebrovascular , may be the most frequent cause of cognitive disorders , particularly in the mild cognitive impairment domain . the cognitive component in question is frontal network systems particularly executive function , attention , and working memory [ 91 , 92 ] . executive interview bedside test ( exit)administration time approximately 1520 min . comprehensive cognitive neurological test in stroke ( coconuts ) . cns vital signs . global tests delis - kaplan executive function system ( dkef ) . wechsler adult intelligence scale ( wais - iv ) components . clinical syndrome orientated , questionnaire based ( 3 ) frontal systems behavior scale ( frsbe ) .(4 ) behavior rating inventory of executive function ( brief ) .(5 ) frontal behavioral inventory ( fbi ) . frontal behavioral inventory ( fbi ) . working memory / executive function tests ( 6 ) trail making tests ( comprehensive , trails a and b , color trails ) .(7 ) letter and category fluency list generation .(8 ) wisconsin card sorting test .(9 ) tower of london test .(10)working memory tests ( verbal and nonverbal ) . trail making tests ( comprehensive , trails a and b , color trails ) . tests of disinhibition / inhibition ( 13)stroop neuropsychological screening test .(14)iowa gambling test . stroop neuropsychological screening test . brown attention deficit disorder scales . depression ( 18)ces - d ( center for epidemiological studies depression scale ) .(19)beck depression scale .(20)hamilton depression inventory . behavioral neurological tests ( 21)faux pas test .(22)reading the mind in the eyes test .(23)hotel task .(24)multiple errands test .(25)ambiguous figures test . other tests that predominantly assess frontal network systems ( 27)visual search and attention test .(28)rey complex figure test . visual search and attention test . qualitative without normative data ( 29)the executive control battery . the executive control battery . a novel approach ( 30)the metacognitive battery incorporating neurological , neuropsychological , and neuropsychiatric syndromes . the metacognitive battery incorporating neurological , neuropsychological , and neuropsychiatric syndromes . aphasia tests useful in motor aphasia , dysnomia , and aphasias in general ( 31)western aphasia battery .(32)boston diagnostic aphasia evaluation . elementary neurological olfaction ( the smell identification test , sensonics gait incontinence primitive reflexes ( grasp reflex , palmomental reflex , sucking reflex ) volitional eye movements . olfaction ( the smell identification test , sensonics primitive reflexes ( grasp reflex , palmomental reflex , sucking reflex ) volitional eye movements . due to the expansive frontal subcortical circuits and their open connections , it may be readily appreciated that the frontal lobes connect with all other regions of the brain including the cerebellum and brainstem . clinical lesion studies have repeatedly shown that no matter where the brain lesion is , whether subcortical gray matter , subcortical white matter , cerebellum , brainstem , or even parietal and occipital lobe , a greater or lesser degree of frontal systems syndrome is present [ 17 , 134 ] . ftd is relatively confined to the frontal and anterior temporal lobes , similarly herpes simplex encephalitis . however , cognitive vascular disorders ( cvd ) , disorders of white matter such as the leukodystrophies and cadasil , vasculitis tbi , ms , most of the toxic metabolic encephalopathic all tend to affect the frontal subcortical networks in a more diffuse fashion and hence present with inattention , dysexecutive syndrome , and dysmemory as the hallmark signature syndromes , considered the most common presentations of frontal network syndromes . many of these will have varying degrees of neuropsychiatric syndrome admixtures most commonly depression and anxiety and perhaps less often disinhibitory behavior , irritability / aggression , obsessive - compulsive disorders , and adult onset adhd . the serotonin toxidrome may present with barely perceptible symptoms to coma with cognitive , somatic , and autonomic manifestations . the cognitive features may include hypomania , agitation , hallucinations , the autonomic hyperthermia , hypertension , tachycardia , diarrhea , and somatic features : myoclonus , tremor , and hyperreflexia . pathophysiologically , there is an increase in cerebral serotonin or overstimulation of the 5ht 2a receptors , often due to maoi 's in combination with ssri , snri , tca , appetite suppressants , or opioids . the diagnosis remains a clinical one . similarly with the neuroleptic malignant syndrome , cognitive changes , autonomic instability , tremors , muscle cramps , tremors , and elevated creatinine phosphokinase the fever is caused by a hypothalamic dopamine receptor blockade and the muscular effects due to blockade of the d2 receptor and the pathophysiology due to low dopamine or dopamine receptor blockade with sympathoadrenal hyperactivity . with the malignant hyperpyrexia syndrome , exposure to anesthetic drugs such as succinylcholine , a neuromuscular blocking agent , halothane , or desflurane , an abnormal muscular activity is induced with hyperpyrexia and circulatory collapse that can be fatal . the inheritance is autosomal dominant , usually for the ryanodine receptor ( gene ryr1 ) located on the sarcoplasmic reticulum and opens in response to an increase in intracellular calcium which is exaggerated in this condition . both have a similar presentation including cognitive autonomic and muscular symptoms and signs but with some important differences such as tachycardia , mydriasis urinary retention , hyperthermia with the former and diarrhea , urination , hypothermia miosis , and bradycardia with the latter . both also present with psychosis , seizures , hallucinations , delirium , and myoclonus . paids ( paroxysmal autonomic instability and dystonia syndrome ) is usually seen after significant traumatic brain injury and also presents with a combination of cognitive alterations , autonomic abnormalities , and , in this instance dystonias rather than muscular rigidity and tremor ( figure 9 ) . frontal syndromes or frontal network syndromes may come to attention by way of the patients symptoms , a clinical syndrome elicited clinically or primarily by neuroimaging findings bearing in mind that this part of the brain has sometimes been termed clinically silent ( tables 35 and figure 10 ) . frontotemporal lobe degeneration as a generic term rather than dementia is recommended because many remain in a category of mci for a long time before frank dementia supervenes . because of the protean manifestations and long duration of decline , sometimes for decades , diagnostic difficulty is the rule . eventually neuroimaging may reveal focal degeneration of frontal insular and temporal lobes although the basal ganglia and spinal motor neurons may also be involved and the presentations may accordingly be initially one of the parkinsonian dementia syndromes ( cbd , psp , and even dsdb ) and mnd . hence it is useful to consider ftld in the context of other primary dementias ( table 6 ) . ftd is more common than ad in those < 60 years and the prevalence is about the same as ad in 6070 group and there are reports of ftd presenting in the third decade . the most common subtype is bv - ftd and a rapidly progressive ftd is seen in association with motor neuron disease with a mean survival of approximately two years . by comparison , the survival of bv - ftd is between 68 years with the language variant subtypes currently demonstrating the longest survival times of 812 years [ 143145 ] . the symptoms and signs initially are subtle , often undiagnosed for years and frequently misdiagnosed as bipolar disease , mania , obsessive compulsive disorder , personality disorder , and depression . dysexecutive function is not specific to ftd and is also seen with other dementias including ad , whatever the emotional impairment is . when the pathology is left sided , language impairment is a major clue and all present with word finding problems . however , in logopenic progressive aphasia ( lpa ) , the person retains the so - called islands of normal speech without dysarthria . the criteria of neary et al . have been augmented by the revised international consensus criteria of rascovsky et al . which are presented in abbreviated form ( appendix 2 ) in . in brief , the major features include ( figure 11 ) the following(i ) progressive deterioration of behavior and/or cognition by observation,(ii ) early behavioral disinhibition,(iii ) early apathy,(iv ) early loss of sympathy or empathy,(v ) early perseverative , stereotyped , or compulsive / ritualistic behavior,(vi ) hyperorality and dietary changes,(vii ) executive deficits with relative sparing of memory and visuospatial functions . progressive deterioration of behavior and/or cognition by observation , early behavioral disinhibition , early loss of sympathy or empathy , early perseverative , stereotyped , or compulsive / ritualistic behavior , hyperorality and dietary changes , executive deficits with relative sparing of memory and visuospatial functions . tar dna binding protein 43 ( tdp-43 ) was identified as the major ubiquitinated protein , with positive inclusions in neurons and glia , occurring in ftd and approximately half of ftd patients have tdp 43 inclusions due to mutations in the progranulin gene found in the frontal and temporal neurons and dentate gyrus . most the remainder is due to tau inclusions caused by mutations in the tau gene . mnd association , sv is mostly linked to tdp 43 , and hippocampal sclerosis ( frequently seen in ftd ) and is usually associated with tdp 43 . about one quarter of progressive lewy body disease patients may also show tdp 43 inclusions [ 148152 ] . overall , in ftd the pathology is , therefore , either a tauopathy or tdp 43 proteinopathy with a small number associated with other pathologies ( table 7 ) . tau is a microtubule - associated protein that is integral to microtubule assembly and in stabilizing microtubules [ 153155 ] . histologically , von economo neurons , a relatively unique spindle - shaped cell found in mammals of such as cetaceans , hominoids , and elephants , are found in the anterior cingulate cortex and fronto - insular regions and are thought to be important in social networks . neurochemically , the presentations of ftd may be due to a significant reduction of serotonin receptors in the cingulate , orbitofrontal , and insular regions . overall , 10% of ftd cases are associated with an autosomal dominant pattern of inheritance with the rest sporadic . both tau and progranulin mutations have been linked to chromosome 17 , and other genetic loci linked to ftd occur on chromosome 3 and 9 . both tau and progranulin mutations cause inclusions in neurons and glia . in general , bv - ftd has the strongest inheritance , sv the less , and pnfa intermediate . ( 1 ) behavioral variant.(2 ) progressive non fluent aphasia ( left perisylvian degeneration associated with tau pathology).(3 ) semantic variant ( anterior temporal degeneration associated with tdp 43 proteinopathy but about 10% have ad).(4 ) logopenic progressive aphasia ( angular gyrus of parietal lobe usually associated with ad pathology ) . progressive non fluent aphasia ( left perisylvian degeneration associated with tau pathology ) . semantic variant ( anterior temporal degeneration associated with tdp 43 proteinopathy but about 10% have ad ) . logopenic progressive aphasia ( angular gyrus of parietal lobe usually associated with ad pathology ) . other neurological deficits may be elicited including peripheral neuropathy , parkinsonism , apraxia , and gaze abnormalities which would support the existence of one of the following syndromes.ftd and parkinsonism and corticobasal - ganglionic syndrome.ftd and als more common with bulbar onset als than limb onset als.ftd and psp present vertical gaze impairment , with early falls axial rigidity . cbd : asymmetric movement disorder with alien hand syndrome and cortical sensory loss . rather than just a primary disorder , cbd may be caused by secondary conditions such as ftd , ad , and creutzfeldt - jakob disease . for example , cbd , psp , and half of people with ftd have mapt ( microtubule associated protein tau ) . ftd and psp present vertical gaze impairment , with early falls axial rigidity . cbd : asymmetric movement disorder with alien hand syndrome and cortical sensory loss . rather than just a primary disorder , cbd may be caused by secondary conditions such as ftd , ad , and creutzfeldt - jakob disease . for example , cbd , psp , and half of people with ftd have mapt ( microtubule associated protein tau ) . it is important to employ specific frontal behavioral batteries such as fbi and frsb as neuropsychological cognitive testing may be normal with a distinct discrepancy in the relative absence of amnesia and visuospatial impairment . structural ( mri / ct ) may reveal frontal or temporal atrophy and functional ( pet , spect ) revealing frontotemporal abnormality much earlier with frontal and/or anterior temporal lobe hypometabolism or hypoperfusion , respectively . a study using mr perfusion scanning revealed similar results and may prove to be more practical . csf tau and ratio of tau to a - beta 42 are significantly lower in ftd than in ad with a sensitivity of 79%90% and specificity of 65% to 97% . in brief , pharmacological management may be employed using serotonergic agents for decreasing obsessions disinhibition , overeating , and repetitive behaviors , as exemplified by the randomized control trial of trazodone . atypical antipsychotics for agitated behavior and avoidance of cholinesterase inhibitors is also recommended as the latter may cause agitation . investigational treatments include both tau- and pgrn- based approaches . for example , inhibition of tau kinases to prevent tau hyperphosphorylation may be accomplished using lithium chloride or valproate . prevention of polyubiquitination to decrease tangle maturation by using hsp-90 and so antagonizing tau fibrillation or stabilizing microtubules using paclitaxel or anti - inflammatory agents is another possible avenue . cognitive impairment no stroke brain at risk stage ( risk factors only : hypertension , diabetes , dyslipidemia ) transient ischemic attacks and cerebral infarct with transient symptoms . brain at risk stage ( risk factors only : hypertension , diabetes , dyslipidemia ) transient ischemic attacks and cerebral infarct with transient symptoms . cognitive impairment subcortical infarct strategic infarct : caudate , thalamus , basal ganglia leukoaraiosis ( figure 11 ) watershed infarction ( figure 12 ) deep venous system tegmentothalamic lesions ( figure 13 ) . strategic infarct : caudate , thalamus , basal ganglia leukoaraiosis ( figure 11 ) watershed infarction ( figure 12 ) deep venous system tegmentothalamic lesions ( figure 13 ) . cortical infarct left angular gyrus ( figure 14 ) , right temporal lobe , frontal . left angular gyrus ( figure 14 ) , right temporal lobe , frontal . cognitive impairment most of the various cerebrovascular syndromes such as small vessel disease , leukoaraiosis , and vasculitis as well as multiple sclerosis and traumatic brain injury affect the brain more diffusely . this is important to consider as many of these patients have a considerably reduced attention span ( and some may be irritable or irascible at the same time due to their disease process ) , and this greatly impacts the mode of testing . extensive neuropsychological testing in such patients is not usually practical and one of the shorter batteries may suffice . however , decline in memory is strongly associated with ad and decline in executive function is strongly associated with cvd . vascular dementia . most of the various cerebrovascular syndromes such as small vessel disease , leukoaraiosis , and vasculitis as well as multiple sclerosis and traumatic brain injury affect the brain more diffusely . this is important to consider as many of these patients have a considerably reduced attention span ( and some may be irritable or irascible at the same time due to their disease process ) , and this greatly impacts the mode of testing . extensive neuropsychological testing in such patients is not usually practical and one of the shorter batteries may suffice . however , decline in memory is strongly associated with ad and decline in executive function is strongly associated with cvd . the origin of cognitive impairment in the context of vascular disease and whether neurodegenerative disease might be implicated ; the following are considered.the pattern and severity of cognitive impairments.the size and location of infarcts including symptomatic infarcts , silent infarcts , and leukoaraiosis.the severity of atrophy patterns in particular hippocampal atrophy and pattern of atrophy of frontal versus parietal.do the vascular lesions adequately explain the cognitive impairment?if the vascular lesions do not adequately explain the cognitive impairment , then neurodegenerative disease is likely present as well . the pattern and severity of cognitive impairments . the size and location of infarcts including symptomatic infarcts , silent infarcts , and leukoaraiosis . the severity of atrophy patterns in particular hippocampal atrophy and pattern of atrophy of frontal versus parietal . if the vascular lesions do not adequately explain the cognitive impairment , then neurodegenerative disease is likely present as well . in studies of neuropsychological series of vascular cognitive impairment and ad , there were no differences in language , constructional abilities , and attention [ 163 , 164 ] . the current definition of dementia is memory centered , requires adl impairment , and does not emphasize the predominant executive dysfunction of the cvd subtypes . for minor cognitive deficits due to vascular disease , the term vci - nd has been recommended ( cognitive deficits that do not meet criteria for dementia but impair minor chores ) . more wm changes or leukoaraiosis and less medial temporal lobe atrophy are seen in cvd when compared to ad although overlap occurs . two types may be discerned ; when the wm lesions are distinct and separated from ventricles , this tends to be more specific for vascular dementia . when the wm hyperintensity is periventricular , this tends to support a neurodegenerative rather than vascular process . a formidable number of conditions can present with white matter hypertensities on mri brain scan ( table 8) . leukoaraiosis may affect one , some , or all of the fsc and quantification will likely become more important in the future ; apart from neuroimaging software capabilities , the junque classification system ( table 9 ) is a very useful manner of quantifying and measuring over time . in general the leukoaraiosis ( la ) on mri can be interpreted as follows.(1 ) mild mri la is correlated with markedly lower scores on episodic memory compared to working memory and is a neuropsychological feature associated with ad.(2 ) moderate mri la correlates with both amnesia and executive dysfunction.(3 ) severe mri la correlates with significantly lower scores on working memory and executive dysfunction . mild mri la is correlated with markedly lower scores on episodic memory compared to working memory and is a neuropsychological feature associated with ad . these findings have treatment implications with the milder forms more amenable to cholinergic therapy and the more severe forms might perform better with dopaminergic therapy . cerebral infarctslacunes microinfarcts up to 5 mmwidening of perivascular spacesincomplete infarctionleukoencephalopathy associated with svd.laminar necrosis selective involvement in the third and fifth layers . granular atrophy patches of gray matter between 2 or 3 arterial territorieslobar hemorrhages linked to a - angiopathy and adsmall hemorrhages association htn at corticosubcortical junction ( slit hemorrhages ) and bg . microinfarcts up to 5 mm widening of perivascular spaces incomplete infarction leukoencephalopathy associated with svd . granular atrophy patches of gray matter between 2 or 3 arterial territories lobar hemorrhages linked to a - angiopathy and ad small hemorrhages leukoaraiosis ( la ) may interrupt the neurotransmitter modulatory systems such as aminergic or corticostriatal and thalamocortical networks . patients with moderate mri la may respond better to aricept especially with junque la scores of 10 compared to those with minimal mri la . there may be at least moderate mri la in context of a dysexecutive syndrome , which may be a marker for the relative preservation of cholinergic neurons . perhaps what is important in this context is that the cerebral vasculature may be observed in real time by fundoscopy . the following were predictive of lacunar stroke : narrower central retinal arterial artery equivalent , wider central retinal vein equivalent , focal arteriolar narrowing , and arteriovenous nicking . overall , a mixed dementia is the most common type ; one that has been aptly termed a vascular tsunami ( hakim ) . population - based autopsy studies indicate that less than 50% of patients have pure ad and many people diagnosed with ad may in fact have vcd . the neuropathology of ad and vci may coexist and influence each other and 8 of 10 of the traditional vascular risk factors also pertain to ad such as hypertension , hyperlipidemia , hyperhomocysteinemia , apoe2 , and apoe 4 . importantly , treatment of vascular risk factors is associated with slower decline in ad with no cvd [ 167 , 171 ] . ad is normally regarded as disease process afflicting the posterior brain , namely , parietotemporal regions with a clinical correlate of dysmemory , visuospatial impairment , geographical disorientation , and only much later behavioral abnormalities that are characteristic of fns . the usual ad variants include a primary progressive aphasia and visuospatial and posterior cortical atrophy syndrome subtypes or variants . reports indicate that about 14%17% of ad patients have nonmemory presentations as an atypical subtype . in addition , executive function is not considered as a component of ad at least in mild - to - moderate disease . however , a subpopulation of ad patients may present with an fns early on including all three principal frontal syndromes of abulia , dysexecutive , and disinhibition . the neurobiology may be a disturbance of the frontal subcortical systems but the underlying etiology remains to be determined . one study found a 10-fold greater neurofibrillary tangle pathology in ba 8 of the frontal lobes . other explanations may include white matter disease , coexistent lewy body disease , and other cerebrovascular pathologies that influence the clinical presentation of the ad as a frontal variant [ 175 , 176 ] . as a pathophysiological process with a few or numerous subcortical plaques , ms is a process that may impair one or more of the fsc 's . recent studies cite a data indicating that 43%72% of patients with ms are considered to have cognitive impairment . ms plaques frequently lie adjacent to the ventricles and so tend to interrupt the long association fibers in the cerebrum such as the fronto - occipital fasciculus , the superior and inferior longitudinal fasciculi . a recent study underscoring the important neurobiology of the frontal subcortical tracts employed frontal neuropsychological as well as behavioral neurological metric tests in comparison to diffusion tensor imaging ( dti ) of the frontal subcortical systems . tests sensitive to the orbitofrontal and cingulate regions of the frontal lobes were used including the hotel task test , iowa gambling test , faux pas test , multiple errands test , and reading the mind in the eyes test . dti evaluations were performed in the frontomedial , frontolateral , orbitofrontal anterior cingulate and a significant difference was found with lower fa values in the fm and fl in the ms patients compared to controls . a significant correlation was also found with loss of fiber integrity in the frontolateral regions and an impairment on the hotel task test and multiple errands test . when cognitive impairment in ms is evaluated , the symptoms elicited are principally inattention , abulia ( apathy ) , dysexecutive syndrome , dysmemory ( working memory ) , and disinhibition ( inappropriate jocularity ) . . stimulant therapy with dopaminergics that pertain to the first four and sodium valproate or carbamazepine is often effective for treating disinhibition syndromes . tbi is associated with a complex syndrome characterized by cognitive ( memory , attention and executive problems ) , elementary neurological symptoms ( headache dizziness , vertigo , imbalance ) , and neuropsychiatric impairment ( anxiety , depression , irritability , irascibility , mania , disinhibition , impulsivity ) . symptoms may be relatively mild to severe and may be present with normal neuroimaging and even anatomical pathology leading to frequent misdiagnosis and under appreciation of the severity of the syndrome . key to understanding the complexity of these syndromes is the current understanding of the pathophysiology ( table 10 ) . the dramatic advances in neuroimaging and alterations in the biochemistry and vascular system might best be described as a networktopathy with neurotransmitter and vascular perturbations that often escape anatomical imaging . the realization that vasospasm is a frequent accompaniment during concussion and tbi has led to the transcranial doppler study initiatives as a novel way of noninvasive monitoring . default mode network imaging may prove to be the most sensitive diagnostic tool yet in diagnosis . the frontal lobes are particularly susceptible to the effects of alcohol , as revealed by recent magnetic resonance spectroscopy studies . in addition , all of the following alcohol related cerebral conditions can affect cognitive functioning in particular frontal systems of working memory , attention , and executive function.alcohol intoxication coma , pathologic intoxication , alcoholic blackouts / memory loss.abstinence or withdrawal syndromes.nutritional disease ( wernicke korsakoff).cerebrovascular infarcts and hemorrhages . cardiomyopathy.cerebellar degeneration.marchiafava-bignami disease.central pontine myelinolysis.alcoholic dementia.cerebral atrophy.neurologic conditions secondary to liver cirrhosis , portal shunts.traumatic brain lesions during intoxication . alcohol intoxication coma , pathologic intoxication , alcoholic blackouts / memory loss . abstinence or withdrawal syndromes . nutritional disease ( wernicke korsakoff ) . traumatic brain lesions during intoxication . previously characterized as a subcortical dementia , this syndrome is still diagnosed by the clinical triad of cognitive , gait impairment and urinary incontinence . the predominant cognitive presentation is a frontal syndrome characterized by dysmemory , dysexecutive syndrome , inattention , and speed of information processing slowing . the frontal lobe syndrome or fns is much more profound than that seen with ad and the memory disturbance is relatively mild with nph , being much more severe with ad . the pathophysiology includes damage to fsc , corpus callosum , thalamus , basal ganglia , and hippocampus . the cognitive impairments respond to appropriate shunt procedures but are usually less so than the improvement in gait impairment . . recently the importance of specific assessment of executive function assessment has been proposed as this can be differentially improved by shunting relative to other cognitive impairments . neuroimaging with pet ( or spect which measures perfusion rather than metabolism ) has been shown to be the best diagnostic tool and a frontal hypometabolism ( in nph ) as opposed to a posterior parieto - occipital hypometabolism ( in ad ) has been shown to differentiate nph from ad better than cisternography . a reappraisal of autoimmune conditions associated with cognitive impairment and at times frank dementia has led to the concept of immunotherapy responsive dementias and encephalopathies . in addition to being treatable and reversible causes of dementia , they may account for up to 20% of the so - called young dementia patients ( < 45 years ) . the cognitive profile includes the usual combination of fsc impairments in speed of information processing , dysmemory , and behavioral abnormalities typically of a fluctuating nature as well as agitation , hallucinations , and seizures . the finding that 1 alpha dendrotoxin antibodies against vgkc noted in patients with morvan 's disease , limbic encephalitis , and neuromyotonia completely changed the understanding from limbic encephalitis being a rare paraneoplastic condition with poor outcome and associated with anti - hu , anti - ma2- or cv2/crmps antibodies . autoimmune dementia syndromes ( aid ) in general are disorders with antibodies against synaptic proteins such as nmda , ampa , and gaba - b receptors and are treatable ( table 11 ) . the identification of a number of neural specific autoantibodies such as voltage gated potassium channel ( vgkc ) antibodies has increased the number of phenotypic presentations of aid . the diagnosis is made by establishing cognitive impairment or an encephalopathic state with clinical , radiologic , or serologic evidence of autoimmune etiology . other causes of dementia require exclusion and a beneficial response to an immunotherapy trial [ 30 , 187 ] . in addition to the specific antibody tests , cerebrospinal fluid analysis is useful with the following parameters regarded as support for aid , raised protein especially over 100 mg / dl , pleocytosis , oligoclonal bands , and igg index elevation . cancer screening is important with computerized chest , abdomen , and pelvis required in all patients , mammography in women and prostate specific antigen and testicular ultrasound in men . treatment options for aid include intravenous methylprednisolone ( 1000 mg 35 days then weekly for 68 weeks ) , ivig if seropositive for gad 65 or ia2 autoantibodies with plasma exchange can not tolerate steroids or ivig . long - term therapy is indicated in responders that may require steroid sparing inhibitor such as azathioprine , mycophenolate , cyclophosphamide , or methotrexate [ 29 , 187 ] . these conditions share several features in that they involve the brain diffusely often with small or microscopic lesions , have relatively covert onset and often subtle signs and syndromes , and may often be overlooked as consequence . the fsc 's are affected as well as the open - ended connections to the posterior parts of the brain , the brainstem , and cerebellum . in addition to standard anatomical brain imaging , csf analysis is required and very often , functional imaging with spect or pet brain scanning , at times magnetic resonance spectroscopy and less often brain biopsy . the challenge is usually in considering or entertaining these disorders in the first place as antibody testing , csf analysis , cerebral catheter angiography and at times brain biopsy as indicated are often the diagnostic . many pathological processes may have as their earliest manifestations some degree of cognitive impairment , most commonly involving the frontal network systems with systemic and meningeal irritation manifestations either absent or appearing much later in the illness . hence frontal network syndromes may be the first sign of a potentially treatable disorder , which if missed , is devastating . with csf analysis of dementia , currently a nonroutine investigation , it is possible to ascribe a slowly dementing state to the most common disorders such as ad or ftd and miss a treatable condition such as chronic cryptococcal meningitis . this has indeed been reported several times and may present a tip of the ice berg phenomenon [ 188190 ] . others seen much less frequently include candida , aspergillus , blastomyces , coccidioides , and histoplasma ( table 12 ) . in the largest series to date ( n = 101 ) of cerebral vasculitis , cognitive impairment in general was present in over three quarters of the patients with altered cognition in 50% and aphasia in 28% of patients . as with other subcortical and more diffuse brain processes , inattention , dysexecutive function , and working memory problems are the most common cognitive disturbances . laboratory ( esr , crp ) , cerebral angiography ( often four vessel catheter cerebral angiography ) and at times brain biopsy are required for diagnosis but the most important clinical error is not to entertain the diagnosis in the first place in the appropriate clinical context ( table 13 ) . conditions that may present with focal or diffuse arterial narrowing and in the differential of vasculitis radiotherapy vasospasm : acute hypertension , migraine , benign reversible cerebral angiopathy , or posterior reversible encephalopathy syndrome ( pres ) lymphoma of the central nervous system intracranial dissection : traumatic , spontaneous , and fibromuscular dysplasia intracranial atherosclerosis recanalizing embolus moyamoya disease and syndrome tumor encasement due to pituitary adenoma or meningioma sickle cell anemia neurofibromatosis [ 192 , 193 ] . vasospasm : acute hypertension , migraine , benign reversible cerebral angiopathy , or posterior reversible encephalopathy syndrome ( pres ) lymphoma of the central nervous system intracranial dissection : traumatic , spontaneous , and fibromuscular dysplasia intracranial atherosclerosis moyamoya disease and syndrome tumor encasement due to pituitary adenoma or meningioma neurofibromatosis [ 192 , 193 ] . in the context of fsc syndromes , the first step is to exclude emergency neurological conditions such as cerebral infarct , hsv-1 , meningitis , subdural hematoma , and mass lesions using anatomical imaging with ct and multimodality mr imaging . thereafter the pursuit of underlying etiological processes is attended often requiring and complemented by functional imaging usually such as dti , f - mri , fluorodeoxyglucose position emission tomography ( fdg - pet ) brain , single - photon emission computed tomography ( spect ) , brain pet pittsburgh compound b ( pib ) , and pet receptor ( dopa ) imaging ( tables 14 and 15 ) . fdg- pet brain in particular has been an important tool in the early diagnosis of mild cognitive impairment ( mci ) and in differentiating types of dementia , with frontotemporal disorders ( ftd ) and alzheimer 's disease ( ad ) . functional imaging is increasingly able to detect pathology , long before the clinical state emerges with pet brain imaging being the most accurate diagnostic method for most common dementia categories . pet brain scan patterns reliably differentiate the major dementia subtypes including the ad variant , posterior cortical atrophy syndrome ( benson syndrome ) , ftd ( frontotemporal hypometabolism ) , and ad ( temporal , parietal , posterior cingulate hypometabolism ) being relatively easily identified . there are also overlap syndromes such as ad and cognitive vascular disorders ( cvd ) , the frontal variant of alzheimer 's disease , and bv - ftd which can not be differentiated easily clinically . other conditions that present predominantly as an fns syndrome include autoimmune dementias , toxic dementias , hiv dementia , and the prefrontal atrophy secondary to chronic stimulation of the pain matrix ( chronic pain syndrome ) [ 198201 ] . positron emission tomography ( pet ) [ 202204 ] and functional magnetic resonance imaging ( fmri ) [ 205 , 206 ] implicate the anterior cingulate cortex ( acc ) and posterior cingulate cortex ( pcc ) having key roles in processing of pain perception . mr perfusion scanning gives similar information to pet brain scanning , and , being based on mri techniques , lack of radiation may give this modality preference in the near future . the default mode network ( dmn ) , for example , reflects the basal or default mode activity of the brain ( without activation procedures ) . regions metabolically active include the posterior cingulate , the precuneus , lateral parietal , lateral temporal , and medial frontal areas . dmn connectivity disruption has been documented in ad , ftd , epilepsy , autism , schizophrenia and depression ( table 16 ) . interestingly , the distribution of the dmn impairment is similar to the fibrillar amyloid deposition seen with ad ( amyloid pet scanning ) . dmn disruption was accurate in identifying major depression with a 94% correct classification with the amygdala , anterior cingulate cortex , parahippocampal gyrus , and hippocampus exhibiting the highest discriminative power in classifying major depression . using fc - mri of the dmn and other rsn such the salience ( for ftd ) , as well as attentional networks , allows rsn patterns to differentiate ad and ftd . in ad , for example , cholinergic ( nicotinic receptors ) and dopaminergic systems measurements have revealed increased c nicotinic binding sites associated with cognitive improvement after rivastigmine for 3 months . in parkinson 's disease using c methyl-4-piperidyl acetate ( mp4a ) , dopaminergic system imaging with f fluorodopa ( fdopa ) showed decreased uptake in the striatum . dti has become the imaging modality of choice to objectively quantify the anatomical pathology which predominantly affects the fiber tracts that occur with traumatic brain injury and multiple sclerosis , for example , often with normal standard mri scans [ 211 , 212 ] . there are a number of pathomechanisms that are associated with brain injury and an understanding of these may lead to avenues of improved brain function after injury . in the endeavor of promoting improvement after brain injury , consideration needs to be given to the following : augmenting and supporting mechanisms of spontaneous recovery , avoiding interventions ( particularly medications ) that may worsen the condition , pharmacotherapy mainly the ascending monoaminergic systems , behavioral therapies using the top down influence of the prefrontal cortex , overcoming inhibitory influences after injury ( mvf - type therapy ) . augmenting and supporting mechanisms of spontaneous recovery , avoiding interventions ( particularly medications ) that may worsen the condition , pharmacotherapy mainly the ascending monoaminergic systems , behavioral therapies using the top down influence of the prefrontal cortex , overcoming inhibitory influences after injury ( mvf - type therapy ) . pharmacotherapy is mainly concerned with the neuromodulatory systems , which are mainly concerned with adjusting signal - to - noise ratios and so influence processing . neuromodulation may be associated with augmenting , diminishing , or prolonging signaling in neuronal networks . there is also a top down regulatory control over the ascending modulatory systems from the pfc to the brainstem neuronal cells groups of ne , da , 5ht , and ach [ 214 , 215 ] . information gleaned mostly from animal models have revealed the cellular and molecular responses to brain injury . currently known processes that are involved in spontaneous recovery in the stroke model , for example , include the following . cellular increased angiogenesis increased synaptogenesis increased dendritic branching and spine density increased neuronal sprouting . increased angiogenesis increased synaptogenesis increased dendritic branching and spine density increased neuronal sprouting . receptor gaba downregulation increased n - methyl - d - aspartate receptor binding . increased n - methyl - d - aspartate receptor binding . molecular increased growth factors increased cell cycle proteins increased growth associate proteins increased inflammatory markers hyperexcitability with long - term potentiation facilitation . increased growth factors increased cell cycle proteins increased growth associate proteins increased inflammatory markers hyperexcitability with long - term potentiation facilitation . future treatment strategies have been proposed for the stroke model but these may be equally applicable to other brain pathologies ( table 17 ) . an important principal revealed by basic science animal models has been the realization that neurotransmitter systems function in phasic as well as tonic modes . this applies to the modulatory ascending systems and correlate with the concept of a bell - shaped curve or the yerkes - dodson inverted u - shaped response seen in animals and humans . this psychological concept relates to the task performance with the horizontal axis representing level of arousal and vertical axis represents a persons particular performance with the peak or top of the bell being the site of optimal performance . this implies that level of monoaminergic function optimal for one particular task may not be so for another task and may be either be sub- or supraoptimal . for example , different levels of norepinephrine ( ne ) may affect different ne receptors , with moderate levels of ne release affecting high affinity alpha 2 a receptors , and even higher ne levels as encountered during stress , involve alpha 1 adrenoreceptors and beta adrenoreceptors . these receptors have opposing functions in the pfc , the former improving and the latter impairing pfc function . the same applies to d1 and d2 receptors where different levels of presynaptic dopamine levels may either improve components of cognition or impair others [ 220 , 221 ] . both ne and da are also considered to have complementary actions affecting cognition function in the pfc as has been reported with respect to spatial wm function , for example . the specific mechanisms of the monoamines of regulation of working memory have implicated the hyperpolarization activated cyclic nucleotide gate cation channels ( hcn channel ) which are localized on heads and necks of dendritic spines near incoming synapses in the superficial layers of monkey pfc . the other functions of the monoamines on the pfc include excitatory and neuroplasticity effects [ 224 , 225 ] . the ne component has been associated with sustained attention when in its phasic mode and distractibility in its tonic mode in nonhuman primates performing a go - no - go visual attentional task . in addition , single unit recordings of the locus coeruleus was associated with optimal performance on a go - no - go visual target detection paradigm and in rhesus monkeys was correlated with phasic firing of ne cells . these include alterations in gene encoding molecules associated with glutamate signaling , cortical development , and the ascending monoaminergic systems as follows . disc 1 ( disrupted in schizophrenia 1 ) : major susceptibility gene for mental illness including schizophrenia , bipolar disease , and depression . disc1 interacts with phosphodiesterase 4b ( pde4b ) and impaired disc 1 function likely leads to overactivation of camp - hcn signaling and weakening of the pfc network connections [ 223 , 227 ] . rgs4 ( regulator of g - protein signaling 4 ) : one of the regulatory proteins acting as gtpase activating proteins that drive g - alpha subunits into inactive gdp form , decreasing their activity . reduced rgs4 leads to an excess of pkc signaling and impaired pfc cognitive function . dgkh ( diacylglycerol kinase isoenzyme ) : one of the lipid kinases catalyzing conversion of diacylglycerol ( dag ) to phosphatidic acid with an overall reduction in dag which is a cofactor in the activation of pkc . loss of dgkh leads to an increase in pkc signaling and mutations are linked to bipolar disease and mania . of note is that the treatments with valproate , lithium , and tamoxifen inhibit pkc signaling . disc 1 ( disrupted in schizophrenia 1 ) : major susceptibility gene for mental illness including schizophrenia , bipolar disease , and depression . disc1 interacts with phosphodiesterase 4b ( pde4b ) and impaired disc 1 function likely leads to overactivation of camp - hcn signaling and weakening of the pfc network connections [ 223 , 227 ] . rgs4 ( regulator of g - protein signaling 4 ) : one of the regulatory proteins acting as gtpase activating proteins that drive g - alpha subunits into inactive gdp form , decreasing their activity . dgkh ( diacylglycerol kinase isoenzyme ) : one of the lipid kinases catalyzing conversion of diacylglycerol ( dag ) to phosphatidic acid with an overall reduction in dag which is a cofactor in the activation of pkc . loss of dgkh leads to an increase in pkc signaling and mutations are linked to bipolar disease and mania . of note is that the treatments with valproate , lithium , and tamoxifen inhibit pkc signaling . dlpfc syndrome and dopaminergic and noradrenergic therapy . using executive function tasks such as word fluency and trails tests , these have been associated with a positive response to clonidine in schizophrenia and korsakoff 's patients [ 232 , 233 ] . medications augmenting da and ne systems have been shown to improve executive function in tourette 's syndrome and attention deficit hyperactivity disorder . a number of medications have been used including tricyclic antidepressants , guanfacine , clonidine , and deprenyl [ 234 , 235 ] . in another example , idazoxan has been correlated with improved executive function in a frontotemporal lobe degeneration patient . based on animal and human data , dopamine agonists such as apomorphine and bromocriptine appear effective in treatment of the condition akinetic mutism spectrum of syndromes , for example . on the other hand , presynaptic dopaminergic agents ( methylphenidate and carbidopa / levodopa ) seem ineffectual [ 237239 ] . midbrain infarction with damage to the dopaminergic neurons also causes akinetic mutism and is responsive to da agonists . in patients where there is damage to the anterior cingulate gyrus with da receptor damage , however , it has been speculated that da agonists may fail . hence loss of dopaminergic input from cortical structures such as the anterior cingulate gyrus as opposed to the striatum may be a factor in determining the type of da treatment . however , other clinical studies in patients with apathy various psychiatric conditions , stroke , wilson 's disease , and hiv dementia have revealed benefit from array of da - ergic medications including bromocriptine , amantadine , methylphenidate , buproprion , and selegiline [ 241245 ] . behavioral disinhibition correlates with a central serotonergic deficiency and serotonergic treatment has been reported to be beneficial in treatment of aggression . one hypothesis regarding aggression is that it may be due to a downregulation of 5-ht2 receptors in the striatum and nucleus accumbens where they occur in abundance [ 247 , 248 ] . serotonergics such as fluoxetine and clomipramine may also be useful in disinhibited , impulsive , aggressive behavior . serenics ( 5ht 1a agonists ) that bind to postsynaptic 5ht 1a receptors have been successful in treating aggression in animals . propranolol , pindolol , and buspirone are examples . in behavioral syndromes that may include mania and noradrenaline overactivity , adrenergic therapy may be beneficial in certain syndromes associated with bilateral inferior orbitofrontal contusions and respond to clonidine . other agents that have been useful include carbamazepine , sodium valproate , propranolol , clonidine , and lithium . aided recently by improved neuroimaging , namely , functional neuroimaging studies have delineated increased activity , either metabolism or blood flow in the orbitofrontal cortex , the head of caudate nucleus , and anterior cingulate gyrus . in general , serotonin reuptake inhibitors as well as clomipramine have been the most advocated pharmacotherapeutic approaches , and right caudate head glucose metabolism ( pet brain scan ) was reduced with successful fluoxetine therapy for obsessive compulsive disorder ( ocd ) . the interaction in cerebral da and 5 ht may account for the improvement seen in some ocd with neuroleptics . in cases where hypometabolism ( pet brain scan ) occurs in the anterior cingulate region and right ofc , this too has been correlated with an improved response to clomipramine therapy . in refractory cases , sumatriptan ( 5ht 1d agonist ) has improved both depression and ocd . both cognitive behavioral therapy and ssri therapy have been shown effective in ocd treatment and their combination potentiated . in a landmark international study , randomized , double - blinded , placebo - controlled trial of inpatient rehabilitation patients ( n = 184 ) with minimally conscious or vegetative state were given amantadine 100 mg bid and increased to 200 mg bid by week 4 . outcome determined by the disability rating scale ( drs ) and recovery was faster in the amantadine group as recorded by 0.24 units difference in the drs per week over the period week 4 to week 16 , in the drs ( p = 0.007 ) . the beneficial effects of amantadine were attributed to presynaptic release facilitation and postsynaptic reuptake blockade thereby augmenting dopaminergic transmission in the mesolimbic , nigrostriatal , and mesocortical circuitry that subserve attention , conation , and arousal . in one of the few randomized - controlled trials , the core frontal component of attention was found to significantly improve speed of information processing in 40 participants with moderate - to - severe tbi receiving methylphenidate at a dose of 0.3 mg / kg twice daily . in a meta - analysis , ftld patients were presumed to have predominant serotonergic deficit as well as dopaminergic deficit with little evidence for ach and ne related impairment . a double blinded , placebo - controlled , crossover trial of trazodone with 300 mg daily revealed a significant improvement using the neuropsychiatric inventory score . trazodone is a selective serotonin reuptake inhibitor with ssri , a 5ht1a , 5ht1c , and 5ht2 , with the active metabolite being a direct serotonin receptor agonist as well as a adrenergic ( alpha 1 , alpha 2 ) and histamine ( h1 ) blocking agent . the effects were noted in the domains of behavior rather than cognition [ 261 , 262 ] . in the flame ( fluoxetine for motor recovery after acute ischemic stroke ) trial patients with ischemic stroke and moderate - to - severe motor deficit , the early use of fluoxetine in combination with physiotherapy enhanced motor recovery at a 3 month evaluation . the mechanism of action is suggested to be a modulation of spontaneous brain plasticity by drugs attributed to brain - derived neurotrophic factor . partly preempted by the advent of the new dsm - v criteria , there is increased emphasis toward a neurobiological models of disease , a renewed neuroimaging focus and using dimensional scales as opposed to categorical diagnoses only ( dsm - iv r ) on traditional neuropsychiatric conditions . the neuropsychiatric disorders include a diverse collection of syndromes affecting behavior , emotion , executive function , and other core frontal network functions that primarily affect emotion , executive function , higher cognition and their circuitry . with a distinct paucity of biomarkers for these syndromes , similar to the approach in neurology , it seems essential to integrate basic neuroscience , neuro - genetics , epigenetics , and neuroradiology in order to establish a foundation for diagnostic based on pathophysiology and presumed etiology . the current psychiatric classifications ( dsm iv ) have had the effect of dichotomizing disease when they are better configured dimensional traits overlapping with normality which is also in accordance with the polygenic mode of inheritance . many patients diagnosed according to the in dsm iv receive multiple diagnoses that is termed comorbidity , probably reflective of a diagnostic artifact due to symptom splitting and lumping . both antipsychotic and antidepressant agents , for example , are used to treat many different psychiatric disorders [ 264 , 265 ] . thinking in terms of symptom clusters and the core components of the frontal subcortical network circuitry may help construct a more neurobiological and pathophysiological relevant approach . this does , however , combine neurological , cerebrovascular , psychiatric , neuropsychological , general medical , and neuroradiological information and consequently the disciplines to advantage ( table 18 ) . serotonergic agents , electroconvulsive therapy , transcranial magnetic stimulation physical exercise , and cognitive behavioral therapy have all been shown to benefit major depressive disorder [ 266268 ] . an important study using pet brain scanning before and after treatment with cbt and a comparison group with the serotonergic agent paroxetine revealed changes in brain metabolism . the cbt group had increased activity in the hippocampal and dorsal cingulum and decreased in the frontal cortex activity whereas the paroxetine group had increased metabolism in the prefrontal cortex and decreased activity in the subgenual cingulate and hippocampus . these findings have been interpreted to suggest that cbt has a top down effect on the medial frontal and cingulate cortex and the pharmacological group work in a bottom up manner [ 269 , 270 ] . interpersonal psychotherapy ( itp ) compared to venlafaxine similarly showed activation of the right posterior cingulate and right basal ganglia and of the right posterior temporal lobe and right basal ganglia in the venlafaxine group ; this time assessed by spect scans . similarly , the psychosurgical treatment , namely , anterior cingulotomy , reserved for severe treatment resistant depression revealed a decrease of metabolism measured by pet brain scanning in the left subgenual pfc and left posterior thalamus , from the preoperative values . stimulants such as methylphenidate increase ne and to a lesser extent da in the pfc while producing lesser effects in the subcortical regions . atomoxetine also increases ne and da with less effect on striatal da and may have a beneficial effect on impulsivity as well . atomoxetine is an important new treatment option for adults with adhd and is particularly so for those who are at risk for substance abuse . atomoxetine is effective and a well - tolerated nonstimulant and the first adhd treatment approved specifically for adult use administered as a single daily dose and is not a controlled substance . . involuntary emotional expression disorder ( ieed ) is a more frequently diagnosed condition especially poststroke , traumatic brain injury , multiple sclerosis , and neurodegenerative diseases . recently , pharmacological treatment has been successfully demonstrated with the efficacy of the dextromethorphan - quinidine combination ( nuedexta ) [ 274 , 275 ] . although in its infancy , one example of a regimen to improve one 's emotional style comprising outlook , self - awareness , attention , resilience , social intuition , and sensitivity to context has been detailed by davidson and begley [ 276278 ] . currently there is support for aphasia therapy , attentional training , rehabilitation of unilateral spatial neglect , and compensatory strategies for apraxia . using information from randomized controlled trials , case series and single case reports to classify recommendations for the following forms of cognitive rehabilitation modalities has been made . this rationale of this mode of therapy proposes that in some patients there is a kind of learned nonuse of the paretic or paralyzed hand or arm after stroke or tbi . a phase iii trial of constraint therapy of 2 weeks of such therapy resulted in significant gains that endured for approximately 2 years . this was subsequently analyzed further to report that comparing early ( 39 months ) and later ( 1521 months ) initiation of cimt after stroke resulted in both groups achieving similar level of significant arm motor function 24 months after enrollment . depending on the pulse frequency , either hypofunction or improved function may ensue due to the inhibitory or excitatory effects on cortical function . this may have application in those instances where inhibitory cortical circuits are operative and if diminished function may return . a randomized trial of treatment - resistant depression with tms has established this as a therapeutic component . in controlled case series studies , mvf has been effective in treating poststroke paresis ( arm or leg ) , phantom limb pain , and complex regional pain syndromeand anxiety . there is also evidence that the modality can modulate pain and reverse objective signs such as inflammation and paralysis [ 283 , 284 ] . patients with arm paresis treated with mvf ( n = 17 , controls n = 19 ) with significant values were reported for hand fim and arm and brunnstrom scores at baseline , 4 weeks and at 6 months . in a study of 40 patients with leg weakness after stroke , compared to best rehabilitation therapy and placebo - controlled with opaque glass , a statistically significant improvement was documented . proposed postulated mechanisms included visuomotor tract restoration leading to an unlearning of the learned paralysis this mechanism may also be attributed to a function of the mirror neuron system that involve interactions between the motor , vision , and proprioception modalities . limb weakness after stroke may be both fiber tract damage , as well as - so - called learned paralysis whereby neurons and their fiber tracts are inhibited and that this can be unlearned using a mirror . also called action observation treatment , this therapy is based on the premise that circuits are activated by observation , similar to those that perform the movement . initial results from observational studies as an add - on therapy appear promising [ 287 , 288 ] .

frontal lobe syndromes , better termed as frontal network systems , are relatively unique in that they may manifest from almost any brain region , due to their widespread connectivity . the understandings of the manifold expressions seen clinically are helped by considering evolutionary origins , the contribution of the state - dependent ascending monoaminergic neurotransmitter systems , and cerebral connectivity . hence , the so - called networktopathies may be a better term for the syndromes encountered clinically . an increasing array of metric tests are becoming available that complement that long standing history of qualitative bedside assessments pioneered by alexander luria , for example . an understanding of the vast panoply of frontal systems ' syndromes has been pivotal in understanding and diagnosing the most common dementia syndrome under the age of 60 , for example , frontotemporal lobe degeneration . new treatment options are also progressively becoming available , with recent evidence of dopaminergic augmentation , for example , being helpful in traumatic brain injury . the latter include not only psychopharmacological options but also device - based therapies including mirror visual feedback therapy .

1. Definition and Synonyms 2. Evolutionary Aspects and Relevance to Clinical Syndromes 3. Neurobiology: The Brain Is a Connectome Consisting of Both Neurochemical Tracts and Macroscopic Hard-Wired Tracts 4. Clinical: Recognizing Primary and 5. Clinical: Diagnostic TestsBeside and Metric 6. Neuropathological States 7. Neuroradiology 8. Treatment options

PMC5253843

gravitational waves , one of the more exotic predictions of einstein s general theory of relativity may , after decades of controversy over their existence , be detected within the next five years . sources such as interacting black holes , coalescing compact binary systems , stellar collapses and pulsars are all possible candidates for detection ; observing signals from them will significantly boost our understanding of the universe . new unexpected sources will almost certainly be found and time will tell what new information such discoveries will bring . gravitational waves are ripples in the curvature of space - time and manifest themselves as fluctuating tidal forces on masses in the path of the wave . the first gravitational - wave detectors were based on the effect of these forces on the fundamental resonant mode of aluminium bars at room temperature . initial instruments were constructed by joseph weber [ 310 , 311 ] and subsequently developed by others . following the lack of confirmed detection of signals , aluminium bar systems operated at and below the temperature of liquid helium were developed [ 253 , 261 , 76 , 170 ] , although work in this area is now subsiding , with only two detectors , auriga and nautilus , continuing to operate . effort also continues to be pursued into cryogenic spherical bar detectors , which are designed to have a wider bandwidth than the cylindrical bars , with the two prototype detectors the dutch minigrail [ 233 , 158 ] and brazilian mrio schenberg [ 159 , 70 ] . however , the most promising design of gravitational - wave detectors , offering the possibility of very high sensitivities over a wide range of frequency , uses widely - separated test masses freely suspended as pendulums on earth or in a drag - free craft in space ; laser interferometry provides a means of sensing the motion of the masses produced as they interact with a gravitational wave . ground - based detectors of this type , based on the pioneering work of forward and colleagues ( hughes aircraft ) , weiss and colleagues ( mit ) , drever and colleagues ( glasgow / caltech ) [ 130 , 129 ] and billing and colleagues ( mpq garching ) , will be used to observe sources whose radiation is emitted at frequencies above a few hz , and space - borne detectors , as originally envisaged by peter bender and jim faller [ 126 , 140 ] at jila , will be developed for implementation at lower frequencies . gravitational - wave detectors of long baseline have been built in a number of places around the world ; in the usa ( ligo project led by a caltech / mit consortium ) [ 45 , 212 ] , in italy ( virgo project , a joint italian / french venture ) [ 61 , 304 ] , in germany ( geo600 project built by a collaboration centred on the university of glasgow , the university of hannover , the max planck institute for quantum optics , the max planck institute for gravitational physics ( albert einstein institute ) , golm and cardiff university ) [ 321 , 151 ] and in japan ( tama300 project ) [ 78 , 294 ] . a space - borne detector , called lisa [ 125 , 217 , 216 ] , was until earlier this year ( 2011 ) under study as a joint esa / nasa mission as one l - class candidate within the esa cosmic visions program ( a recent meeting detailing these missions can be found here ) . funding constraints within the us now mean that esa must examine the possibility of flying an l - class mission with european - only funding . the official esa statement on the next steps for lisa can be found here . when completed , this detector array would have the capability of detecting gravitational wave signals from violent astrophysical events in the universe , providing unique information on testing aspects of general relativity and opening up a new field of astronomy . it is also possible to observe the tidal effects of a passing gravitational wave by doppler tracking of separated objects . for example , doppler tracking of spacecraft allows the earth and an interplanetary spacecraft to be used as test masses , where their relative positions can be monitored by comparing the nearly monochromatic microwave signal sent from a ground station with the coherently returned signal sent from the spacecraft . by comparing these signals , a doppler frequency time series /0 , where 0 is the central frequency from the ground station , can be generated . peculiar characteristics within the doppler time series , caused by the passing of gravitational waves , can be studied in the approximate frequency band of 10 to 0.1 hz . several attempts have been made in recent decades to collect such data ( ulysses , mars observer , galileo , mars global surveyor , cassini ) with broadband frequency sensitivities reaching 10 ( see for a thorough review of gravitational - wave searches using doppler tracking ) . there are currently no plans for dedicated experiments using this technique ; however , incorporating doppler tracking into another planetary mission would provide a complimentary precursor mission before dedicated experiments such as lisa are launched . the technique of doppler tracking to search for gravitational - wave signals can also be performed using pulsar - timing experiments . millisecond pulsars are known to be very precise clocks , which allows the effects of a passing gravitational wave to be observed through the modulation in the time of arrival of pulses from the pulsar . many noise sources exist and , for this reason , it is necessary to monitor a large array of pulsars over a long observation time . further details on the techniques used and upper limits that have been set with pulsar timing experiments can be found from groups such as the european pulsar timing array , the north american nanohertz observatory for gravitational waves [ 190 , 191 ] , and the parkes pulsar timing array . all the above detection methods cover over 13 orders of magnitude in frequency ( see figure 1 ) equivalent to covering from radio waves to x - rays in the electromagnetic spectrum . figure 1the sensitivity of various gravitational - wave detection techniques across 13 orders of magnitude in frequency . at the low frequency end the sensitivity curves for pulsar timing arrays ( based on current observations and future observations with the square kilometre array ) are extrapolated from figure 4 in . in the mid - range lisa , decigo and bbo are described in more detail in section 7 , with the decigo and bbo sensitivity curves taken from models given in . at the high frequency the sensitivities are represented by three generations of laser interferometers : ligo , advanced ligo and the einstein telescope ( see sections 6 , 6.3.1 and 6.3.2 ) . also included is a representative sensitivity for the auriga , allegro and nautilus bar detectors . the sensitivity of various gravitational - wave detection techniques across 13 orders of magnitude in frequency . at the low frequency end the sensitivity curves for pulsar timing arrays ( based on current observations and future observations with the square kilometre array ) are extrapolated from figure 4 in . in the mid - range lisa , decigo and bbo are described in more detail in section 7 , with the decigo and bbo sensitivity curves taken from models given in . at the high frequency the sensitivities are represented by three generations of laser interferometers : ligo , advanced ligo and the einstein telescope ( see sections 6 , 6.3.1 and 6.3.2 ) . also included is a representative sensitivity for the auriga , allegro and nautilus bar detectors . for a popular account of the development of the gravitational - wave field the reader should consult chapter 10 of black holes and time warps by kip s. thorne , or the more recent books , einstein s unfinished symphony , by marcia bartusiak and gravity from the ground up , by bernard schutz . a comprehensive review of developments toward laser interferometer detectors is found in fundamentals of interferometric gravitational wave detectors by peter saulson , and discussions relevant to the technology of both bar and interferometric detectors are found in the detection of gravitational waves edited by david blair . in addition to the wealth of articles that can be found on the home site of this journal , there are also various informative websites that can easily be found , including the homepages of the various international collaborative projects searching for gravitational waves , such as the ligo scientific collaboration . some early relativists were sceptical about the existence of gravitational waves ; however , the 1993 nobel prize in physics was awarded to hulse and taylor for their experimental observations and subsequent interpretations of the evolution of the orbit of the binary pulsar psr 1913 + 16 [ 185 , 295 ] , the decay of the binary orbit being consistent with angular momentum and energy being carried away from this system by gravitational waves . gravitational waves are produced when matter is accelerated in an asymmetrical way ; but due to the nature of the gravitational interaction , detectable levels of radiation are produced only when very large masses are accelerated in very strong gravitational fields . such a situation can not be found on earth but is found in a variety of astrophysical systems . gravitational wave signals are expected over a wide range of frequencies ; from 10 hz in the case of ripples in the cosmological background to 10 hz from the formation of neutron stars in supernova explosions . the most predictable sources are binary star systems . however , there are many sources of much - greater astrophysical interest associated with black - hole interactions and coalescences , neutron - star coalescences , neutron stars in low - mass x - ray binaries such as sco - x1 , stellar collapses to neutron stars and black holes ( supernova explosions ) , pulsars , and the physics of the early universe . for a full discussion of sources partly because observation of the velocity and polarisation states of the signals will allow a direct experimental check of the wave predictions of general relativity ; but , more importantly , because the detection of the signals should provide observers with new and unique information about astrophysical processes . it is interesting to note that the gravitational wave signal from a coalescing compact binary star system has a relatively simple form and the distance to the source can be obtained from a combination of its signal strength and its evolution in time . if the redshift at that distance is found , hubble s constant the value for which has been a source of lively debate for many years may then be determined with , potentially , a high degree of accuracy [ 282 , 180 ] . only now are detectors being built with the technology required to achieve the sensitivity to observe such interesting sources . gravitational waves are most simply thought of as ripples in the curvature of space - time , their effect being to change the separation of adjacent masses on earth or in space ; this tidal effect is the basis of all present detectors . gravitational wave strengths are characterised by the gravitational - wave amplitude h , given by 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h = { { 2\delta l } \over l},$$\end{document } where l is the change in separation of two masses a distance l apart ; for the strongest - allowed component of gravitational radiation , the value of h is proportional to the third time derivative of the quadrupole moment of the source of the radiation and inversely proportional to the distance to the source . the radiation field itself is quadrupole in nature and this shows up in the pattern of the interaction of the waves with matter . the problem for the experimental physicist is that the predicted magnitudes of the amplitudes or strains in space in the vicinity of the earth caused by gravitational waves even from the most violent astrophysical events are extremely small , of the order of 10 or lower [ 273 , 272 ] . indeed , current theoretical models on the event rate and strength of such events suggest that in order to detect a few events per year from coalescing neutron - star binary systems , for example , an amplitude sensitivity close to 10 over timescales as short as a millisecond is required . if the fourier transform of a likely signal is considered it is found that the energy of the signal is distributed over a frequency range or bandwidth , which is approximately equal to 1/timescale . for timescales of a millisecond the bandwidth is approximately 1000 hz , and in this case the spectral density of the amplitude sensitivity is obtained by dividing 10 by the square root of 1000 . thus , detector noise levels must have an amplitude spectral density lower than 10 hz over the frequency range of the signal . signal strengths at the earth , integrated over appropriate time intervals , for a number of sources are shown in figure 2 . the weakness of the signal means that limiting noise sources like the thermal motion of molecules in the critical components of the detector ( thermal noise ) , seismic or other mechanical disturbances , and noise associated with the detector readout , whether electronic or optical , must be reduced to an extremely low level . for signals above 10 hz ground based experiments are possible , but for lower frequencies where local fluctuating gravitational gradients and seismic noise on earth become a problem , it is best to consider developing detectors for operation in space . the earliest experiments in the field were ground based and were carried out by joseph weber of the university of maryland in the 1960s . with colleagues he began by looking for evidence of excitation of the normal modes of the earth by very low frequency gravitational waves . efforts to detect gravitational waves via the excitation of earth s normal modes was also pursued by weiss and block . weber then moved on to look for tidal strains in aluminium bars , which were at room temperature and were well isolated from ground vibrations and acoustic noise in the laboratory [ 310 , 311 ] . the bars were resonant at 1600 hz , a frequency where the energy spectrum of the signals from collapsing stars was predicted to peak . despite the fact that weber observed coincident excitations of his detectors placed up to 1000 km apart , at a rate of approximately one event per day , his results were not substantiated by similar experiments carried out in several other laboratories in the usa , germany , britain and russia . it seems unlikely that weber was observing gravitational - wave signals because , although his detectors were very sensitive , being able to detect strains of the order of 10 over millisecond timescales , their sensitivity was far away from what was predicted to be required theoretically . development of weber bar type detectors continued with significant emphasis on cooling to reduce the noise levels , although work in this area is now subsiding with efforts continuing on auriga , nautilus , minigrail [ 233 , 158 ] and mario schenberg [ 159 , 70 ] . in around 2003 , the sensitivity of km - scale interferometric gravitational - wave detectors began to surpass the peak sensitivity of these cryogenic bar detectors ( 10 ) and , for example , the ligo detectors reached their design sensitivities at almost all frequencies by 2005 ( peak sensitivity 2 10 at 200 hz ) , see section 6.1 for more information on science runs of the recent generation of detectors . in addition to gaining better strain sensitivities , interferometric detectors have a marked advantage over resonant bars by being sensitive to a broader range of frequencies , whereas resonant bar are inherently sensitive only to signals that have significant spectral energy in a narrow band around their resonant frequency . the concept and design of gravitational - wave detectors based on laser interferometers will be introduced in the following section 3.2 . an interferometric design of gravitational - wave detector offers the possibility of very high sensitivities over a wide range of frequency . it uses test masses , which are widely separated and freely suspended as pendulums to isolate against seismic noise and reduce the effects of thermal noise ; laser interferometry provides a means of sensing the motion of these masses produced as they interact with a gravitational wave ( figure 3 ) . this technique is based on the michelson interferometer and is particularly suited to the detection of gravitational waves as they have a quadrupole nature . waves propagating perpendicular to the plane of the interferometer will result in one arm of the interferometer being increased in length while the other arm is decreased and vice versa . the induced change in the length of the interferometer arms results in a small change in the intensity of the light observed at the interferometer output . as will be explained in detail in the next section 4 , the sensitivity of an interferometric gravitational - wave detector is limited by noise from various sources . taking this frequency - dependent noise floor into account for example , the design sensitivity for initial ligo is show in figure 4 plotted alongside the achieved sensitivities of the three individual interferometers during the fifth science run ( see section 6.1 ) . such strain sensitivities are expected to allow a reasonable probability for detecting gravitational wave sources . however , in order to guarantee the observation of a full range of sources and to initiate gravitational - wave astronomy , a sensitivity or noise performance approximately ten times better in the mid - frequency range and several orders of magnitude better at 10 hz , is desired . therefore , initial detectors will be upgraded to an advanced configuration , such as advanced ligo , which will be ready for operation around 2015 . figure 4measured sensitivity of the initial ligo interferometers during the s5 science run ( see section 6.1.2 ) . reproduced with permission from . measured sensitivity of the initial ligo interferometers during the s5 science run ( see section 6.1.2 ) . reproduced with permission from . for the initial interferometric detectors , a noise floor in strain close to 2 10 hz was achieved . detecting a strain in space at this level implies measuring a residual motion of each of the test masses of about 8 10 m / hz over part of the operating range of the detector , which may be from 10 hz to a few khz . this sets a formidable goal for the optical detection system at the output of the interferometer . in this section we discuss the main noise sources , which limit the sensitivity of interferometric gravitational - wave detectors . fundamentally it should be possible to build systems using laser interferometry to monitor strains in space , which are limited only by the heisenberg uncertainty principle ; however there are other practical issues , which must be taken into account . fluctuating gravitational gradients pose one limitation to the interferometer sensitivity achievable at low frequencies , and it is the level of noise from this source , which dictates that experiments to look for sub - hz gravitational - wave signals have to be carried out in space [ 291 , 275 , 93 , 184 ] . in general , for ground - based detectors the most important limitations to sensitivity result from the effects of seismic and other ground - borne mechanical noise , thermal noise associated with the test masses and their suspensions , and quantum noise , which appears at high frequency as shot noise in the photocurrent from the photodiode , which detects the interference pattern and can appear at low frequency as radiation pressure noise due to momentum transfer to the test masses from the photons when using high laser powers . the significance of each of these sources will be briefly reviewed . seismic noise at a reasonably quiet site on the earth follows a spectrum in all three dimensions close to 10f m / hz ( where here and elsewhere we measure f in hz ) and thus if the disturbance to each test mass must be less than 3 10 m / hz at , for example , 30 hz , then the reduction of seismic noise required at that frequency in the horizontal direction is greater than 10 . since there is liable to be some coupling of vertical noise through to the horizontal axis , along which the gravitational - wave - induced strains are to be sensed , a significant level of isolation has to be provided in the vertical direction also . isolation can be provided in a relatively simple way by making use of the fact that , for a simple pendulum system , the transfer function to the pendulum mass of the horizontal motion of the suspension point falls off as 1/(frequency ) above the pendulum resonance . in a similar way isolation can be achieved in the vertical direction by suspending a mass on a spring . in the case of the virgo detector system the design allows operation to below 10 hz and here a seven - stage horizontal pendulum arrangement is adopted with six of the upper stages being suspended with cantilever springs to provide vertical isolation , with similar systems developed in australia and at caltech . for the geo600 detector , where operation down to 50 hz was planned , a triple pendulum system is used with the first two stages being hung from cantilever springs to provide the vertical isolation necessary to achieve the desired performance . this arrangement is then hung from a plate mounted on passive rubber isolation mounts and on an active ( electro - mechanical ) anti - vibration system [ 257 , 298 ] . the upgraded seismic isolation for the total isolation will be provided by one external stage ( hydraulics ) , two stages of in - vacuum active isolation , and being completed by the test mass suspensions [ 55 , 166 ] . for clarity , the two stages of in - vacuum isolation are shown in figure 5 , whereas the test - mass suspensions are shown separately in figure 6 . figure 5internal stages of the large chamber seismic isolation system for advanced ligo ( image is inverted for clarity ) . figure 6cad drawing of quad suspension system for advanced ligo , showing the mirror test mass at the bottom and where the uppermost section is attached to the third stage platform of the large chamber seismic isolation system shown in figure 5 . internal stages of the large chamber seismic isolation system for advanced ligo ( image is inverted for clarity ) . cad drawing of quad suspension system for advanced ligo , showing the mirror test mass at the bottom and where the uppermost section is attached to the third stage platform of the large chamber seismic isolation system shown in figure 5 . in order to cut down motion at the pendulum frequencies , active damping of the pendulum modes has to be incorporated , and to reduce excess motion at low frequencies around the micro - seismic peak , low - frequency isolators have to be incorporated . these low - frequency isolators can take different forms tall inverted pendulums in the horizontal direction and cantilever springs whose stiffness is reduced by means of attractive forces between magnets for the vertical direction in the case of the virgo system , scott russell mechanical linkages in the horizontal and torsion bar arrangements in the vertical for an australian design , and a seismometer / actuator ( active ) system as shown here for advanced ligo and also used in geo600 . such schemes can provide sufficiently - large reduction in the direct mechanical coupling of seismic noise through to the suspended mirror optic to allow operation down to 3 hz [ 98 , 134 ] . however , it is also possible for this vibrational seismic noise to couple to the suspended optic through the gravitational field . gravity gradients , caused by direct gravitational coupling of mass density fluctuations to the suspended mirrors , were identified as a potential source of noise in ground - based gravitational - wave detectors in 1972 . the noise associated with gravity gradients was first formulated by saulson and spero , with later developments by hughes and thorne and cella and cuoco . these studies suggest that the dominant source of gravity gradients arise from seismic surface waves , where density fluctuations of the earth s surface are produced near the location of the individual interferometer test masses , as shown in figure 7 . figure 7time - lapsed schematic illustrating the fluctuating gravitational force on a suspended mass by the propagation of a surface wave through the ground . time - lapsed schematic illustrating the fluctuating gravitational force on a suspended mass by the propagation of a surface wave through the ground . the magnitude of the rms motion of the interferometer test masses , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\tilde x(\omega)$\end{document } , can be shown to be 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde x(\omega ) = { { 4\pi g\rho } \over { { \omega ^2}}}\beta ( \omega)\tilde w(\omega),$$\end{document } where is the earth s density near the test mass , g is newton s constant , is the angular frequency of the seismic spectrum , ( ) ; is a dimensionless reduced transfer function that takes into account the correlated motion of the interferometer test masses in addition to the reduction due to the separation between the test mass and the earth s surface , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\tilde w(\omega)$\end{document } is the displacement rms - averaged over 3-dimensional directions . in order to eliminate noise arising from gravity gradients , a detector would have to be operated far from these density fluctuations , that is , in space . . however , there are two proposed approaches for reducing the level of gravity - gradient noise in future ground - based detectors . a monitor and subtraction method can be used , where an array of seismometers can be distributed strategically around each test mass to monitor the relevant ground motion ( and ground compression ) that would be expected to couple through local gravity . a subtraction signal may be developed from knowing how the observed density fluctuations couple to the motion of each test mass , and can potentially allow a significant reduction in gravity - gradient noise . another approach is to choose a very quiet location , or better still , to also go underground , as is already going ahead for lcgt . since the dominant source of gravity - gradient noise is expected to arise from surface waves on the earth , the observed gravity - gradient noise will decrease with depth into the earth . current estimates suggest that gravity - gradient noise can be suppressed down to around 1 hz by careful site selection and going 150 m underground . the most promising approach ( or likely only approach ) to detecting gravitational waves whose frequency is below 1 hz is to build an interferometer in space . thermal noise associated with the mirror masses and the last stage of their suspensions is the most significant noise source at the low frequency end of the operating range of initial long baseline gravitational wave detectors . advanced detector configurations are also expected to be limited by thermal noise at their most sensitive frequency band [ 211 , 238 , 168 , 121 ] . the thermal noise in the operating range comes from the tails of these resonant modes . for any simple harmonic oscillator such as a mass hung on a spring or hung as a pendulum , the spectral density of thermal motion of the mass can be expressed as 3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${x^2}(\omega ) = { { 4{k_{\rm{b}}}t\omega _ 0 ^ 2\phi ( \omega ) } \over { \omega m[{{(\omega _ 0 ^ 2 - { \omega ^2})}^2 } + \omega _ 0 ^ 4{\phi ^2}(\omega)]}},$$\end{document } where kb is boltzmann s constant , t is the temperature , m is the mass and ( ) is the loss angle or loss factor of the oscillator of angular resonant frequency 0 . this loss factor is the phase lag angle between the displacement of the mass and any force applied to the mass at a frequency well below 0 . in the case of a mass on a spring , the loss factor is a measure of the mechanical loss associated with the material of the spring . for a pendulum , thus , the loss factor is lower than that of the material , which is used for the wires or fibres used to suspend the pendulum . indeed , following saulson it can be shown that for a pendulum of mass m , suspended on four wires or fibres of length l , the loss factor of the pendulum is related to the loss factor of the material by 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\phi _ { { \rm{pend}}}}(\omega ) = { \phi _ { { \rm{mat}}}}(\omega){{4\sqrt { tei } } \over { mgl}},$$\end{document } where i is the moment of the cross - section of each wire , and t is the tension in each wire , whose material has a young s modulus e. in general , for most materials , it appears that the intrinsic loss factor is essentially independent of frequency over the range of interest for gravitational - wave detectors ( although care has to be taken with some materials in that a form of damping known as thermo - elastic damping can become important for wires of small cross - section and for some bulk crystalline materials ) . in order to estimate the internal thermal noise of a test mass , each resonant mode of the mass when the detector operating range is well below the resonances of the masses , following saulson , the effective spectral density of thermal displacement of the front face of each mass can be expressed as the summation of the motion of the various mechanical resonances of the mirror as also discussed by gillespie and raab . however , this intuitive approach to calculating the thermally - driven motion is only valid when the mechanical loss is distributed homogeneously and , therefore , not valid for real test - mass mirrors . the mechanical loss is known to be inhomogeneous due to , for example , the localisation of structural defects and stress within the bulk material , and the mechanical loss associated with the polished surfaces is higher than the levels typically associated with bulk effects . therefore , levin suggested using a direct application of the fluctuation - dissipation theorem to the optically - sensed position of the mirror substrate surface . this technique imposes a notional pressure ( of the same spatial profile as the intensity of the sensing laser beam ) to the front face of the substrate and calculates the resulting power dissipated in the substrate on its elastic deformation under the applied pressure . using such an approach we find that sx(f ) can then be described by the relation 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_x}(f ) = { { 2{k_{\rm{b}}}t } \over { { \pi ^2}{f^2}}}{{{w_{{\rm{diss } } } } } \over { f_0 ^ 2}},$$\end{document } where f0 is the peak amplitude of the notional oscillatory force and wdiss is the power dissipated in the mirror described as , 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${w_{{\rm{diss } } } } = \omega \int { \epsilon ( r)\phi ( r)\partial v } , $ $ \end{document } where (r ) and (r ) are the strain and mechanical loss located at specific positions within the volume v. this formalisation highlights the importance of where mechanical dissipation is located with respect to the sensing laser beam . in particular , the thermal noise associated with the multi - layer dielectric mirror coatings , required for high reflectivity , will in fact limit the sensitivity of second - generation gravitational - wave detectors at their most sensitive frequency band , despite these coatings typically being only 4.5 m in thickness . identifying coating materials with lower mechanical loss , and trying to understand the sources of mechanical loss in existing coating materials , is a major r&d effort targeted at enhancements to advanced detectors and for third generation instruments . in order to keep thermal noise as low as possible the mechanical loss factors of the masses and pendulum resonances further , the test masses must have a shape such that the frequencies of the internal resonances are kept as high as possible , must be large enough to accommodate the laser beam spot without excess diffraction losses , and must be massive enough to keep the fluctuations due to radiation pressure at an acceptable level . test masses currently range in mass from 6 kg for geo600 to 40 kg for advanced ligo . to approach the best levels of sensitivity discussed earlier the loss factors of the test masses must be 3 10 or lower , and the loss factor of the pendulum resonances should be smaller than 10 . obtaining these values puts significant constraints on the choice of material for the test masses and their suspending fibres . geo600 utilises very - low - loss silica suspensions , a technology , which should allow detector sensitivities to approach the level desired for second generation instruments [ 105 , 266 , 268 ] , since the intrinsic loss factors in samples of synthetic fused silica have been measured down to around 5 10 . still , the use of other materials such as sapphire is being seriously considered for future detectors [ 104 , 195 , 266 ] such as in lcgt [ 234 , 247 ] . the technique of hydroxy - catalysis bonding provides a method of jointing oxide materials in a suitably low - loss way to allow monolithic suspension systems to be constructed . a recent discussion on the level of mechanical loss and the associated thermal noise in advanced detectors resulting from hydroxy - catalysis bonds is given by cunningham et al . . images of the geo600 monolithic mirror suspension and of the prototype advanced ligo mirror suspension are shown in figure 8 . figure 8monolithic silica suspension of ( a ) geo600 6 kg mirror test mass suspended from 4 fibres of thickness 250 m and ( b ) prototype monolithic suspension for advanced ligo at lasti ( mirror mass of 40 kg , silica fibre thickness 400 m ) . monolithic silica suspension of ( a ) geo600 6 kg mirror test mass suspended from 4 fibres of thickness 250 m and ( b ) prototype monolithic suspension for advanced ligo at lasti ( mirror mass of 40 kg , silica fibre thickness 400 m ) . for gravitational - wave signals to be detected , the output of the interferometer must be held at one of a number of possible points on an interference fringe . an obvious point to choose is halfway up a fringe since the change in photon number produced by a given differential change in arm length is greatest at this point ( in practice this is not at all a sensible option and interferometers are operated at , or near , a dark fringe see sections 5.1 and 5.4 ) . the interferometer may be stabilised to this point by sensing any changes in intensity at the interferometer output with a photodiode and feeding the resulting signal back , with suitable phase , to a transducer capable of changing the position of one of the interferometer mirrors . information about changes in the length of the interferometer arms can then be obtained by monitoring the signal fed back to the transducer . as mentioned earlier , it is very important that the system used for sensing the optical fringe movement on the output of the interferometer can resolve strains in space of 2 10 hz or lower , or differences in the lengths of the two arms of less than 10 m / hz , a minute displacement compared to the wavelength of light 10 m. a limitation to the sensitivity of the optical readout scheme is set by shot noise in the detected photocurrent . from consideration of the number of photoelectrons ( assumed to obey poisson statistics ) measured in a time it can be shown that the detectable strain sensitivity depends on the level of laser power p of wavelength used to illuminate the interferometer of arm length l , and on the time , such that : 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{detectable}}\;{\rm{strain}}\;{\rm{in}}\;{\rm{time}}\;\tau = { 1 \over l}{\left [ { { { \lambda hc } \over { 2{\pi ^2}p\tau } } } \right]^{1/2}},$$\end{document } or 8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{detectable}}\;{\rm{strain}}\;{{\rm{(hz)}}^{- 1/2 } } = { 1 \over l}{\left [ { { { \lambda hc } \over { { \pi ^2}p } } } \right]^{1/2}},$$\end{document } where c is the velocity of light , h is planck s constant and we assume that the photodetectors have a quantum efficiency 1 . thus , achievement of the required strain sensitivity level requires a laser , operating at a wavelength of 10 m , to provide 6 10 power at the input to a simple michelson interferometer . this is a formidable requirement ; however , there are a number of techniques which allow a large reduction in this power requirement and these will be discussed in section 5 . as the effective laser power in the arms is increased , another phenomenon becomes increasingly important arising from the effect on the test masses of fluctuations in the radiation pressure . one interpretation on the origin of this radiation pressure noise may be attributed to the statistical uncertainty in how the beamsplitter divides up the photons of laser light . each photon is scattered independently and therefore produces an anti - correlated binomial distribution in the number of photons , n , in each arm , resulting in a \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$ \propto \sqrt n$\end{document } fluctuating force from the radiation pressure . this is more formally described as arising from the vacuum ( zero - point ) fluctuations in the amplitude component of the electromagnetic field . this additional light entering through the dark - port side of the beamsplitter , when being of suitable phase , will increase the intensity of laser light in one arm , while decreasing the intensity in the other arm , again resulting in anti - correlated variations in light intensity in each arm [ 109 , 110 ] . the laser light is essentially in a noiseless coherent state as it splits at the beamsplitter and fluctuations arise entirely from the addition of these vacuum fluctuations entering the unused port of the beamsplitter . using this understanding of the coherent state of the laser , shot noise arises from the uncertainty in the phase component ( quadrature ) of the interferometer s laser field and is observed in the quantum fluctuations in the number of detected photons at the interferometer output . radiation pressure noise arises from uncertainty in the amplitude component ( quadrature ) of the interferometer s laser field . both result in an uncertainty in measured mirror positions . for the case of a simple michelson , shown in figure 3 , the power spectral density of the fluctuating motion of each test mass m resulting from fluctuation in the radiation pressure at angular frequency is given by , 9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { x^2}(\omega ) = \left({{{4ph } \over { { m^2}{\omega ^4}c\lambda } } } \right),$$\end{document } where h is planck s constant , c is the speed of light and is the wavelength of the laser light . in the case of an interferometer with fabry - prot cavities , where the typical number of reflections is 50 , displacement noise x due to radiation pressure fluctuations scales linearly with the number of reflections , such that , 10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { x^2}(\omega ) = { 50 ^ 2 } \times \left({{{4ph } \over { { m^2}{\omega ^4}c\lambda } } } \right).$$\end{document } radiation pressure may be a significant limitation at low frequency and is expected to be the dominant noise source in advanced ligo between around 10 and 50 hz . of course the effects of the radiation pressure fluctuations can be reduced by increasing the mass of the mirrors , or by decreasing the laser power at the expense of degrading sensitivity at higher frequencies . since the effect of photoelectron shot noise decreases when increasing the laser power as the radiation pressure noise increases , a fundamental limit to displacement sensitivity is set . for a particular frequency of operation , there will be an optimum laser power within the interferometer , which minimises the effect of these two sources of optical noise , which are assumed to be uncorrelated . this sensitivity limit is known as the standard quantum limit ( sql ) and corresponds to the heisenberg uncertainty principle , in its position and momentum formulation ; see [ 133 , 109 , 110 , 221 ] . firstly , it is possible to reach the sql at a tuned range of frequencies , when dominated by either radiation - pressure noise or shot noise , by altering the noise distribution in the two quadratures of the vacuum field . there are a number of proposed designs for achieving this in future interferometric detectors , such as a squeezed - input interferometer [ 110 , 300 ] , a variational - output interferometer or a squeezed - variational interferometer using a combination of both techniques . this technique may be of importance in allowing an interferometer to reach the sql at a particular frequency , for example , when using lower levels of laser power and otherwise being dominated by shot noise . experiments are under way to incorporate squeezed - state injection as part of the upgrades to current gravitational - wave detectors , and where a squeezing injection bench has already been installed in the geo600 gravitational - wave detector , which expects to be able to achieve an up - to-6 db reduction in shot noise using the current interferometer configuration . similar experiments are also under way to demonstrate variational readout , where ponderomotive squeezing arises from the naturally - occurring correlation of radiation - pressure noise to shot noise upon reflection of light from a mirror [ 117 , 271 ] secondly , if correlations exist between the radiation - pressure noise and the shot - noise displacement limits , then it is possible to bypass the sql , at least in principle . there are at least two ways by which such correlations may be introduced into an interferometer . one scheme is where an optical cavity is constructed , where there is a strong optical spring effect , coupling the optical field to the mechanical system . this is already the case for the geo600 detector , where the addition of a signal recycling cavity creates such correlation , where signal recycling is described in section 5.2 . other schemes of optical springs have been studied , such as optical bars and optical levers [ 103 , 101 ] . another method is to use suitable filtering at optical frequencies of the output signal , by means of long fabry - prot cavities , which effectively introduces correlation [ 201 , 116 ] . as explained in section 4.4.1 , high - power laser light is needed to overcome limitations of a detector s sensitivity due to photoelectron shot noise . the situation can be helped greatly if a multi - pass arrangement is used in the arms of the interferometer as this multiplies up the apparent movement by the number of bounces the light makes in the arms . the multiple beams can either be separate , as in an optical delay line [ 313 , 95 ] , or may lie on top of each other as in a fabry - prot resonant cavity , as shown in figure 9 . figure 9michelson interferometers with ( a ) delay lines and ( b ) fabry - prot cavities in the arms of the interferometer . michelson interferometers with ( a ) delay lines and ( b ) fabry - prot cavities in the arms of the interferometer . optimally , the light should be stored for a time comparable to the characteristic timescale of the signal . thus , if signals of characteristic timescale 1 msec are to be searched for , the number of bounces should be approximately 50 for an arm length of 3 km . with 50 bounces the required laser power it can be shown that an optimum signal - to - noise ratio in a michelson interferometer can be obtained when the arm lengths are such that the output light is very close to a minimum ( this is not intuitively obvious and is discussed more fully in ) . thus , rather than lock the interferometer to the side of a fringe as discussed above in section 4.4.1 , it is usual to make use of a modulation technique to operate the interferometer close to a null in the interference pattern . an electro - optic phase modulator placed in front of the interferometer can be used to phase modulate the input laser light . if the arms of the interferometer are arranged to have a slight mismatch in length this results in a detected signal , which , when demodulated , is zero with the cavity exactly on a null fringe and changes sign on different sides of the null providing a bipolar error signal ; this can be fed back to the transducer controlling the interferometer mirror to hold the interferometer locked near to a null fringe ( this is the rf readout scheme discussed in section 5.4 ) . in this situation , if the mirrors are of very low optical loss , nearly all of the light supplied to the interferometer is reflected back towards the laser . in other words the impedance matching can be improved by placing another mirror of correctly chosen transmission a power recycling mirror between the laser and the interferometer so that a resonant cavity is formed between this mirror and the rest of the interferometer ; in the case of perfect impedance matching , no light is reflected back towards the laser [ 131 , 278 ] . there is then a power build - up inside the interferometer as shown in figure 10 . this can be high enough to create the required kilowatts of laser light at the beamsplitter , starting from an input laser light of only about 10 w. figure 10the implementation of power recycling on a michelson interferometer with fabry - prot cavities . the implementation of power recycling on a michelson interferometer with fabry - prot cavities . to be more precise , if the main optical power losses are those associated with the test mass mirrors taken to be a per reflection the intensity inside the whole system considered as one large cavity is increased by a factor given by ( l)/(ca ) , where the number of bounces , or light storage time , is optimised for signals of timescale and the other symbols have their usual meaning . then : 11\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\rm{detectable}}\;{\rm{strain}}\;{\rm{in}}\;{\rm{time}}\;\tau = { \left [ { { { \lambda ha } \over { 4\pi lp{\tau ^2 } } } } \right]^{1/2}}.$$\end{document } to enhance further the sensitivity of an interferometric detector and to allow some narrowing of the detection bandwidth , which may be valuable in searches for continuous wave sources of gravitational radiation , another technique known as signal recycling can be implemented [ 231 , 292 , 169 ] . this relies on the fact that sidebands created on the light by gravitational - wave signals interacting with the arms do not interfere destructively and so do appear at the output of the interferometer . if a mirror of suitably - chosen reflectivity is put at the output of the system as shown in figure 11 , then the sidebands can be recycled back into the interferometer , where they resonate , and hence the signal size over a given bandwidth ( set by the mirror reflectivity ) is enhanced . figure 11the implementation of signal recycling on a michelson interferometer with fabry - prot cavities . the centre of this frequency band is set by the precise length of the cavity formed by the signal recycling mirror and the cavities in the interferometer arms . thus , control of the precise position of the signal recycling mirror allows tuning of the frequency at which the performance is peaked . often signal recycling will be used to provide a narrow bandwidth to search for continuous wave sources as mentioned above , however it may also be used with a relatively broad bandwidth , centred away from zero frequency , and this application is useful for matching the frequency response of the detector to expected spectral densities of certain broadband or chirping signals . using appropriate optical configurations that employ power and signal recycling as described in sections 5.1 and 5.2 , the required laser power may thus be reduced to a level ( in the range of 10 to 100 w ) where laser sources are now available ; however stringent requirements on technical noise must be satisfied . power fluctuations as described above in section 5.1 gravitational - wave interferometers are typically designed to operate with the length of the interferometer arms set such that the output of the interferometer is close to a minimum in the output light . if the interferometer were operated exactly at the null point in the fringe pattern , then , in principle , it would be insensitive to power fluctuations in the input laser light . however in practice there will be small offsets from the null position causing some sensitivity to this noise source . in this case , it can be shown that the required power stability of the laser in the frequency range of interest for gravitational - wave detection may be estimated to be 12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta p } \over p } \simeq h{(\delta l / l)^{- 1}},$$\end{document } where p / p are the relative power fluctuations of the laser , and l is the offset from the null fringe position for an interferometer of arm length l. from calculations of the effects of low - frequency seismic noise for the initial designs of long baseline detectors it can be estimated that the rms motion will be on the order of 10 m when the system is operating . taking strains of around 3 10 hz at 300 hz , as is the case for advanced ligo , requires power fluctuations of the laser not to exceed 13\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta p } \over p } \leq { 10^{- 7}}{\rm{h}}{{\rm{z}}^{- 1/2}}\;\;{\rm{at } } \simeq 300\;{\rm{hz}}.$$\end{document } to achieve this level of power fluctuation typically requires the use of active stabilisation techniques of the type developed for argon ion lasers . however , it should be noted that the most stringent constraint on laser - power fluctuations in future ground - based detectors , where circulating laser powers will approach 1 mw or even beyond , will ultimately arise from classical radiation pressure on the mirrors , as described in section 4.4.2 . frequency fluctuations for a simple michelson interferometer it can be shown that a change x in the differential path length , x , of the interferometer arms causes a phase change in the light at the interferometer output given by = ( 2/c ) ( vx + xv ) where is a change in the laser frequency and c is the speed of light . it follows that if the lengths of the interferometer arms are exactly equal ( i.e. , x = 0 ) , the interferometer output is insensitive to fluctuations in the frequency of the input laser light , provided that , in the case of fabry - prot cavities in the arms , the fluctuations are not so great that the cavities can not remain on resonance . however , in practice , differences in the optical properties of the interferometer mirrors result in slightly different effective arm lengths , a difference of perhaps a few tens of metres . then the relationship between the limit to detectable gravitational - wave amplitude and the fluctuations d of the laser frequency is given by 14\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta \nu } \over \nu } \simeq h{(x / l)^{- 1}}.$$\end{document } hence , to achieve the target sensitivity used in the above calculation using a detector with arms of length 4 km , maximal fractional frequency fluctuations of , 15\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta \nu } \over \nu } \leq { 10^{- 21}}{\rm{h}}{{\rm{z}}^{- 1/2}},$$\end{document } are required . this level of frequency noise may be achieved by the use of appropriate laser - frequency stabilisation systems involving high finesse reference cavities .although the calculation here is for a simple michelson interferometer , similar arguments apply to the more sophisticated systems with arm cavities , power recycling and signal recycling discussed earlier and lead to the same conclusions . frequency stabilisation is also important in other applications such as in high resolution optical spectroscopy , optical frequency standards [ 223 , 312 ] and fundamental quantum measurements . the best reference cavities , such as those developed by the ye and hnsch groups , have reported a frequency stability performance of around 10 , which is broadly equivalent to that achieved for ground - based gravitational - wave detectors when scaling by cavity length . beam geometry fluctuations fluctuations in the lateral or angular position of the input laser beam , along with changes in its size and variations in its phase - front curvature may all couple into the output signal of an interferometer and reduce its sensitivity . these fluctuations may be due to intrinsic laser mechanical noise ( from water cooling for example ) or from seismic motion of the laser with respect to the isolated test masses . as an example of their importance , fluctuations in the lateral position of the beam may couple into interferometer measurements through a misalignment of the beamsplitter with respect to the interferometer mirrors . a lateral movement z of the beam incident on the beamsplitter , coupled with an angular misalignment of the beamsplitter of /2 results in a phase mismatch of the interfering beams , such that 16\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \phi = ( 4\pi /\lambda)\alpha \delta z.$$\end{document } a typical beamsplitter misalignment of 10 radians means that to achieve sensitivities of the level described above using a detector with 3 or 4 km arms , and 50 bounces of the light in each arm , a level of beam geometry fluctuations at the beamsplitter of close to 10 m / hz at 300 hz is required.typically , and ignoring possible ameliorating effects of the power recycling cavity on beam geometry fluctuations , this will mean that the beam positional fluctuations of the laser need to be suppressed by several orders of magnitude . the two main methods of reducing beam geometry fluctuations are 1 ) passing the input beam through a single mode optical fibre and 2 ) using a resonant cavity as a mode cleaner [ 270 , 288 , 317 , 83].passing the beam through a single mode optical fibre helps to eliminate beam geometry fluctuations , as deviations of the beam from a gaussian tem00 mode are equivalent to higher - order spatial modes , which are thus attenuated by the optical fibre . however , there are limitations to the use of optical fibres , mainly due to the limited power - handling capacity of the fibres ; care must also be taken to avoid introducing extra beam geometry fluctuations from movements of the fibre itself.a cavity may be used to reduce beam geometry fluctuations if it is adjusted to be resonant only for the tem00 mode of the input light . the use of a resonant cavity should allow the handling of higher laser powers and has the additional benefits of acting as a filter for fast fluctuations in laser frequency and power [ 288 , 317 ] . this latter property is extremely useful for the conditioning of the light from some laser sources as will be discussed below . as described above in section 5.1 gravitational - wave interferometers are typically designed to operate with the length of the interferometer arms set such that the output of the interferometer is close to a minimum in the output light . if the interferometer were operated exactly at the null point in the fringe pattern , then , in principle , it would be insensitive to power fluctuations in the input laser light . however in practice there will be small offsets from the null position causing some sensitivity to this noise source . in this case , it can be shown that the required power stability of the laser in the frequency range of interest for gravitational - wave detection may be estimated to be 12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta p } \over p } \simeq h{(\delta l / l)^{- 1}},$$\end{document } where p / p are the relative power fluctuations of the laser , and l is the offset from the null fringe position for an interferometer of arm length l. from calculations of the effects of low - frequency seismic noise for the initial designs of long baseline detectors it can be estimated that the rms motion will be on the order of 10 m when the system is operating . taking strains of around 3 10 hz at 300 hz , as is the case for advanced ligo , requires power fluctuations of the laser not to exceed 13\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta p } \over p } \leq { 10^{- 7}}{\rm{h}}{{\rm{z}}^{- 1/2}}\;\;{\rm{at } } \simeq 300\;{\rm{hz}}.$$\end{document } to achieve this level of power fluctuation typically requires the use of active stabilisation techniques of the type developed for argon ion lasers . however , it should be noted that the most stringent constraint on laser - power fluctuations in future ground - based detectors , where circulating laser powers will approach 1 mw or even beyond , will ultimately arise from classical radiation pressure on the mirrors , as described in section 4.4.2 . frequency fluctuations for a simple michelson interferometer it can be shown that a change x in the differential path length , x , of the interferometer arms causes a phase change in the light at the interferometer output given by = ( 2/c ) ( vx + xv ) where is a change in the laser frequency and c is the speed of light . it follows that if the lengths of the interferometer arms are exactly equal ( i.e. , x = 0 ) , the interferometer output is insensitive to fluctuations in the frequency of the input laser light , provided that , in the case of fabry - prot cavities in the arms , the fluctuations are not so great that the cavities can not remain on resonance . however , in practice , differences in the optical properties of the interferometer mirrors result in slightly different effective arm lengths , a difference of perhaps a few tens of metres . then the relationship between the limit to detectable gravitational - wave amplitude and the fluctuations d of the laser frequency is given by 14\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta \nu } \over \nu } \simeq h{(x / l)^{- 1}}.$$\end{document } hence , to achieve the target sensitivity used in the above calculation using a detector with arms of length 4 km , maximal fractional frequency fluctuations of , 15\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\delta \nu } \over \nu } \leq { 10^{- 21}}{\rm{h}}{{\rm{z}}^{- 1/2}},$$\end{document } are required . this level of frequency noise may be achieved by the use of appropriate laser - frequency stabilisation systems involving high finesse reference cavities . although the calculation here is for a simple michelson interferometer , similar arguments apply to the more sophisticated systems with arm cavities , power recycling and signal recycling discussed earlier and lead to the same conclusions . frequency stabilisation is also important in other applications such as in high resolution optical spectroscopy , optical frequency standards [ 223 , 312 ] and fundamental quantum measurements . the best reference cavities , such as those developed by the ye and hnsch groups , have reported a frequency stability performance of around 10 , which is broadly equivalent to that achieved for ground - based gravitational - wave detectors when scaling by cavity length . beam geometry fluctuations fluctuations in the lateral or angular position of the input laser beam , along with changes in its size and variations in its phase - front curvature may all couple into the output signal of an interferometer and reduce its sensitivity . these fluctuations may be due to intrinsic laser mechanical noise ( from water cooling for example ) or from seismic motion of the laser with respect to the isolated test masses . as an example of their importance , fluctuations in the lateral position of the beam may couple into interferometer measurements through a misalignment of the beamsplitter with respect to the interferometer mirrors . a lateral movement z of the beam incident on the beamsplitter , coupled with an angular misalignment of the beamsplitter of /2 results in a phase mismatch of the interfering beams , such that 16\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta \phi = ( 4\pi /\lambda)\alpha \delta z.$$\end{document } a typical beamsplitter misalignment of 10 radians means that to achieve sensitivities of the level described above using a detector with 3 or 4 km arms , and 50 bounces of the light in each arm , a level of beam geometry fluctuations at the beamsplitter of close to 10 m / hz at 300 hz is required . typically , and ignoring possible ameliorating effects of the power recycling cavity on beam geometry fluctuations , this will mean that the beam positional fluctuations of the laser need to be suppressed by several orders of magnitude . the two main methods of reducing beam geometry fluctuations are 1 ) passing the input beam through a single mode optical fibre and 2 ) using a resonant cavity as a mode cleaner [ 270 , 288 , 317 , 83 ] . passing the beam through a single mode optical fibre helps to eliminate beam geometry fluctuations , as deviations of the beam from a gaussian tem00 mode are equivalent to higher - order spatial modes , which are thus attenuated by the optical fibre . however , there are limitations to the use of optical fibres , mainly due to the limited power - handling capacity of the fibres ; care must also be taken to avoid introducing extra beam geometry fluctuations from movements of the fibre itself . a cavity may be used to reduce beam geometry fluctuations if it is adjusted to be resonant only for the tem00 mode of the input light . the use of a resonant cavity should allow the handling of higher laser powers and has the additional benefits of acting as a filter for fast fluctuations in laser frequency and power [ 288 , 317 ] . this latter property is extremely useful for the conditioning of the light from some laser sources as will be discussed below . from equation ( 7 ) it can be seen that the photon - noise limited sensitivity of an interferometer is proportional to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt p$\end{document } where p is the laser power incident on the interferometer , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt \lambda$\end{document } where is the wavelength of the laser light . thus , single frequency lasers of high output power and short wavelength are desirable . with these constraints in mind , argon - ion lasers emitting light at 514 nm were used to illuminate several interferometric gravitational - wave detector prototypes , see , for example , [ 287 , 264 ] . however , their efficiency , reliability , controllability and noise performance has ruled them out as suitable laser sources for current and future gravitational wave detectors . nd : yag lasers , emitting at 1064 nm or frequency doubled to 532 nm , present an alternative . the longer ( infrared ) wavelength may initially appear less desirable than the 514 nm of the argon - laser and the frequency doubled 532 nm , as more laser power is needed to obtain the same sensitivity . in addition , the resulting increase in beam diameter leads to a need for larger optical components . for example in an optical cavity the diameter of the beam at any point is proportional to the square root of the wavelength and to keep diffractive losses at each test mass below 1 10 , it can be shown that the diameter of each test mass must be greater than 2.6 times the beam diameter at the test mass . thus , the test masses for gravitational wave detectors have to be 1.4 times larger in diameter for infrared than for green light . however , nd : yag sources at 1064 nm have demonstrated some compelling advantages , in particular the demonstration of scaling the power up to levels suitable for second generation interferometers ( 200 w ) combined with their superior efficiency [ 286 , 306 , 200 ] . frequency - doubled nd : yvo lasers at 532 nm have currently only been demonstrated to powers approaching 20 w and have not been actively stabilised to the levels needed for gravitational - wave detectors . an additional problem associated with shorter wavelength operation is the potential for increased absorption , possibly leading to photochemistry ( damage ) in the coating materials , in addition to increased scatter . for this reason , all the initial long - baseline interferometer projects , along with their respective upgrades , have chosen some form of nd : yag light source at 1064 nm . as an example , the laser power is being upgraded from 10 w in initial ligo to 180 w for advanced ligo to improve the snr of the shot - noise - limited regime . this power will be delivered by a three stage injection - locked oscillator scheme [ 120 , 237 , 157 , 144 ] . the first stage uses a monolithic non - planar ring oscillator ( npro ) to initially produce 2 w of output power . this output is subsequently amplified by a four - head nd : yvo laser amplifier to a power of 35 w , which is in turn delivered into an injection locked nd : yag oscillator to produce 200 w of output power . other laser developments are being pursued , such as high - power - fibre lasers , which are currently being investigated by the aei in germany and prototyped for advanced virgo by grverie et al . in france . fibre amplifiers show great potential for extrapolation to higher laser powers in addition to lower production costs . third - generation interferometric gravitational - wave detectors , such as the einstein telescope , require input laser powers of around 500 w at 1064 nm in order to achieve their high - frequency shot - noise - limited sensitivities . low - frequency sensitivity is expected to be achieved through the use of separate low - power interferometers with silicon optics operating at cryogenic temperatures [ 269 , 262 ] . longer wavelengths are proposed here due to excessive absorption in silicon at 1064 nm and the expected low absorption ( less than 0.1 ppm / cm ) at around 1550 nm . worldwide laser developments may provide new baseline light sources that can provide different wavelength and power options for future detectors . however , the stringent requirements on the temporal and spatial stability for gravitational - wave detectors are beyond that sought in other laser applications . therefore , a dedicated laser - development program will be required to continue beyond the second - generation interferometers in order to design and build a laser system that meets third - generation requirements , as discussed in more detail in . another key area of laser development , targeted at improving the sensitivity of future gravitational - wave detectors , is in the use of special optical modes to reduce thermal noise . it can be shown that the amplitude of thermal noise associated with the mirror coatings is inversely proportional to the beam radius . the configuration within current interferometers is designed to inject and circulate tem00 optical modes , which have a gaussian beam profile . to keep diffraction losses suitably low for this case ( < 1 ppm ) , a beam radius of a maximum size 35% of the radius of the test mass mirror can be used . the thermal noise could be further reduced if optical modes are circulated that have a larger effective area , yet not increasing the level of diffraction losses . this would be possible through the use of higher - order laguerre - gauss beams , and other a more in - depth discussion of how these optical schemes can be implemented and the potential increase in detector sensitivity attainable can be found in . despite the very - low levels of optical absorption in fused silica at 1064 nm , thermal loading due to high - levels of circulating laser powers within advanced gravitational - wave detectors will cause significant thermal loading . in the case of advanced ligo , thermal lensing will be most significant in the input test masses of the fabry - prot cavities , where the beam must transmit through the substrate in addition to the high - power within the cavity being incident on the coating surfaces . thermal distortion in the optics will be sensed by hartmann sensors and coupled to two schemes of thermal compensation . firstly , ring resistance heaters will be installed around the barrel of the input mass in order to compensate for the beam heating the central region of the optics , as demonstrated for radius of curvature tuning in geo600 . secondly , a flexible co2 laser based system will be used to deposit heat onto the reaction mass ( otherwise called the compensation plate ) for the input test mass , as demonstrated in initial ligo [ 209 , 308 ] . the laser beam shape and intensity can be easily modified from outside with the vacuum system and can therefore adapt to non - uniformities in the absorption and other changes in the interferometer s thermal state . it should also be noted that energy can couple from the optical modes resonating in the interferometer fabry - prot cavities and the acoustic modes of the test masses . when there is sufficient coupling between these optical and mechanical modes , and the mechanical modes have a suitably high - quality factor , then mechanical resonances can be rung - up by the large circulating laser power to the point where the interferometer is no longer stable , a phenomenon called parametric instabilities . mechanical dampers that are tuned to damp at high - frequency yet not significantly increasing thermal noise at low - frequency are being considered in possible upgrades to advanced detectors , in addition to other schemes , such as active feedback to damp problematic modes provided through the electrostatic actuators . there are various schemes that can be applied to readout the gravitational - wave signal from an interferometer . if the interferometer laser has a frequency of f1 then a passing gravitational wave , with frequency fgw , will introduce sidebands onto the laser with a frequency of fs = f1 fgw . a readout scheme must be able to decouple the gravitational - wave component , with frequencies of order 100 hz , from the far higher laser frequency at hundreds of tera - hz . to do this it needs to be able to compare the sideband frequency with a known stable optical local oscillator . ideally this oscillator would be the laser light itself ( a homodyne scheme ) , but the initial generation of gravitational - wave detectors are operated at a dark fringe ( i.e. , the interferometer is held , so as the light from the arms completely destructively interferes at the beam splitter ) , so no light at the laser frequency exits ( a gravitational wave will alter the arms lengths and constructively interfere , causing light to exit , but only at the sideband frequency ) . the standard scheme used by the initial interferometers is a radio frequency ( rf ) heterodyne readout . in this case the laser light is modulated at an rf ( called schnupp modulation ) prior to entering the interferometer arms , giving rise to sidebands offset from the laser frequency at the rf . the interferometer is set up to allow these rf sidebands to exit at the output port . this can be used as a local optical oscillator with which to demodulate the gravitational wave sidebands . however , the demodulation will introduce a beat between the rf and the gravitational wave frequency , which must be removed by a second ( hence heterodyne ) demodulation at the rf . the preferred method for future detectors is a dc scheme ( see [ 147 , 309 , 177 ] for motivations and advantages of using such a scheme ) . in this instead the interferometer is held just off the dark fringe , so some light at the laser frequency reaches the output to serve as the local oscillator . prior to the start of the 21st century there existed several prototype laser interferometric detectors , constructed by various research groups around the world at the max - planck - institt fr quantenoptik in garching , at the university of glasgow , at the california institute of technology , at the massachusetts institute of technology , at the institute of space and astronautical science in tokyo and at the astronomical observatory in tokyo . these detectors had arm lengths varying from 10 m to 100 m and had either multibeam delay lines or resonant fabry - prot cavities in their arms . the 10 m detector that used to exist at glasgow figure 12the 10 m prototype gravitational wave detector at glasgow . the 10 m prototype gravitational wave detector at glasgow . the sensitivities of some of these detectors reached a level better than 10 for millisecond bursts such that the technology could be considered sufficiently mature to propose the construction of detectors of much longer baseline that would be capable of reaching the performance required to have a real possibility of detecting gravitational waves . an international network of such long baseline gravitational wave detectors has now been constructed and commissioned , and science - quality data from these has been produced and analysed since 2002 ( see section 6.1 and section 6.2 for a review of recent science data runs and results ) . the american ligo project comprises two detector systems with arms of 4 km length , one in hanford , washington , and one in livingston , louisiana ( also known as the ligo hanford observatory 4k [ lho 4k ] and ligo livingston observatory 4k [ llo 4k ] , or h1 and l1 , respectively ) . one half length , 2 km , interferometer was also contained inside the same evacuated enclosure at hanford ( also known as the lho 2k , or h2 ) . the design goal of the 4 km interferometers was to have a peak strain sensitivity between 100200 hz of 3 10 hz ( see figure 15 ) , which was achieved during the fifth science run ( section 6.1 ) . a birds - eye view of the hanford site showing the central building and the directions of the two arms is shown in figure 13 . in october 2010 the ligo detectors shut down and decommissioning began in preparation for the installation of a more sensitive instrument known as advanced ligo ( see section 6.3.1 ) . figure 13a bird s eye view of the ligo detector , sited in hanford , washington . a bird s eye view of the ligo detector , sited in hanford , washington . the french / italian virgo project comprises a single 3 km arm - length detector at cascina near pisa . as mentioned earlier , it is designed to have better performance than the other detectors , down to 10 hz . the tama300 detector , which has arms of length 300 m , at the tokyo astronomical observatory was the first of the beyond - prototype detectors to become operational . this detector is built mainly underground and partly has the aim of adding to the gravitational - wave detector network for sensitivity to events within the local group of galaxies , but is primarily a test bed for developing techniques for future larger - scale detectors . initial operation of the interferometer was achieved in 1999 and power recycling was implemented for data taking in 2003 . all the systems mentioned above are designed to use resonant cavities in the arms of the detectors and use standard wire - sling techniques for suspending the test masses . the german / british detector , geo600 , built near hannover , germany , is somewhat different . it makes use of a four - pass delay - line system with advanced optical signal - enhancement techniques , utilises very - low loss - fused silica suspensions for the test masses , and , despite its smaller size , was designed to have a sensitivity at frequencies above a few hundred hz comparable to the first phases of virgo and ligo during their initial operation . it uses both power recycling ( section 5.1 ) and tunable signal recycling ( section 5.2 ) , often referred to together as dual recycling . to gain the most out of the detectors as a true network , the ligo and geo600 teams signed a memorandum of understanding ( mou ) , under the auspices of the ligo scientific collaboration ( lsc ) , allowing complete data sharing between the two groups . part of this agreement has been to ensure that both ligo and geo600 have taken data in coincidence ( see below ) . coincident data taking , and joint analysis , has also occurred between the tama300 project and the lsc detectors . the virgo collaboration also signed an mou with the lsc , which has allowed data sharing since may 2006 . the operation and commissioning of these detectors is a continually - evolving process , and the current state of this review only covers developments until late-2010 . for the most up - to - date information on detectors readers are advised to consult the proceedings of the amaldi meetings , gwdaw / gwpaw ( gravitational wave data analysis workshops ) , and gwadw ( gravitational wave advanced detectors workshops ) see for a list of past conferences . for the first and second generations of detector , much effort has gone into estimating the expected number of sources that might be observable given their design sensitivities . in particular , for what are thought to be the strongest sources : the coalescence of neutron - star binaries or black holes ( see section 6.2.2 for current rates as constrained by observations ) . these estimates , based on observation and simulation , are summarised in table 5 of and the realistic rates suggest initial detectors would expect to see 0.02 , 0.004 and 0.007 events per year for neutron - star - binary , black - hole - neutron - star , and black - hole - binary systems , respectively ( it should be noted that there is a range of possible rates consistent with current observations and models ) . second generation detectors ( see section 6.3.1 ) , which can observe approximately 1000 time more volume than the initial detectors might , expect to see 40 , 10 , and 20 per year for the same sources . with such rates a great deal of astrophysics could be possible the commissioning and improvement of the various gravitational - wave detectors has been suspended at various stages to take data for astrophysical analysis . these have been times when it was considered that the detectors were sensitive and stable enough ( or had made sufficient improvements over earlier states ) to make astrophysical searches worthwhile . within the lsc these have been called the science ( s ) runs , for virgo they have been the virgo science runs ( vsr ) , and for tama300 they have been the data taking ( dt ) periods . a time - line of science runs for the various interferometric detectors , can be seen in figure 14 . figure 14a time - line of the science runs of the first generation interferometric gravitational - wave detectors , from their first lock to mid-2011 . a time - line of the science runs of the first generation interferometric gravitational - wave detectors , from their first lock to mid-2011 . a figure of merit for the sensitivity of a detector is to calculate its horizon distance . this is the maximum range out to which it could see the coalescence of two 1.4 m neutron stars that are optimally oriented and located ( i.e. , with the orbital plane perpendicular to the line - of - sight , and with this plane parallel to the detector plane , so that the antenna response is at its maximum ) at a signal - to - noise ratio of 8 . the horizon distance can be converted to a range that is an average over all sky locations and source orientations ( i.e. not the best case scenario ) by dividing it by 2.26 ) we shall use this angle averaged range throughout the rest of this review . the first interferometric detector to start regular data taking with sufficient sensitivity and stability to enable it to potentially detect gravitational waves from the the galactic centre was tama300 . over the period between august 1999 to january 2004 tama had nine data - taking periods ( denominated dt1 - 9 ) over which time its typical strain noise sensitivity , in its most sensitive frequency band improved from 3 10 hz to 1.5 10 hz . tama300 operated in coincidence with the ligo and geo600 detectors for two of the science data - taking periods . more recently focus has shifted to the cryogenic laser interferometer observatory ( clio ) prototype detector [ 324 , 164 ] , designed to test technologies for a future second - generation japanese detector called the large - scale cryogenic gravitational - wave telescope ( lcgt ) ( see section 6.3.1 ) . the first ligo detector to achieve lock ( meaning having the interferometer stably held on a dark fringe of the interference pattern , with light resonating throughout the cavity ) all three detectors had achieved lock and have since undergone many periods of commissioning and science data taking . over the period from mid-2001 to mid-2002 the commissioning process improved the detectors peak sensitivities by several orders of magnitude , with l1 going from 10 10 hz at 150 hz . in summer 2002 it was decided that the detectors were at a sensitivity , and had a good enough lock stability , to allow a science data - taking run . from 23 august to 9 september 2002 the three ligo detectors , along with geo600 ( and , for some time , tama300 ) , undertook their first coincident science run , denoted s1 ( see for the state of the ligo and geo600 detectors at the time of s1 ) . at this time the most sensitive detector was l1 with a peak sensitivity at around 300 hz of 23 10 hz . the best strain amplitude sensitivity curve for s1 ( and the subsequent ligo science runs ) can be seen in figure 15 . the amount of time over the run that the detectors were said to be in science mode , i.e. , stable and with the interferometer locked , called their duty cycle , or duty factor , was 42% for l1 , 58% for h1 and 73% for h2 . for the most sensitive detector , l1 , the inspiral range was typically 0.08 mpc . the s6 curve is preliminary and based on h(t ) data that has not been completely reviewed and may be subject to change . the s6 curve is preliminary and based on h(t ) data that has not been completely reviewed and may be subject to change . for the second science run ( s2 ) , from 14 february to 14 april 2003 , the noise floor was considerably improved over s1 by several upgrades including : improving and stabilising the optical levers used to measure the mirror orientation to reduce the low frequency ( 50 hz ) noise ; replacing the coil drivers that are used as actuators to control the position and orientation of suspended mirrors , to improve the mid - frequency ( 50200 hz ) noise floor ; and increasing the laser power in the interferometer to reduce shot noise and improve the high frequency ( 200 hz ) sensitivity ( see section iia of for a more thorough description of the detector improvements made for s2 ) . these changes improved the sensitivities by about an order of magnitude across the frequency band with a best strain , for l1 , of 3 10 hz between 200300 hz . the duty factor during s2 was 74% for h1 , 58% for h2 and 38% for l1 , with a triple coincidence time when all three detectors were in lock of 22% of the run . the average inspiral ranges during the run were approximately 0.9 , 0.4 and 0.3 mpc for l1 , h1 and h2 respectively . for the the third science run ( s3 ) , from 31 october 2003 to 9 january 2004 , the detectors were again improved , with the majority of sensitivity increase in the mid - frequency range . the best sensitivity , which was for h1 , was 5 10 hz between 100200 hz . the duty factors were 69% for h1 , 63% for h2 and only 22% for l1 , with a 16% triple coincidence time . l1 s poor duty factor was due to large levels of anthropogenic seismic noise near the site during the day . the fourth science run ( s4 ) , from 22 february to 23 march 2005 , saw less - drastic improvements in detector sensitivity across a wide frequency band , but did make large improvements for frequencies 70 hz . between s3 and s4 a better seismic isolation system , which actively measured and countered for ground motion , was installed in l1 , greatly reducing the amount of time it was thrown out of lock . for h1 the laser power was able to be increased to its full design power of 10 w . the duty factors were 80% for h1 , 81% for h2 and 74% for l1 , with a 56% triple coincidence time . by mid - to - late 2005 the detectors had equaled their design sensitivities over most of the frequency band and were also maintaining good stability and high duty factors . it was decided to perform a long science run with the aim of collecting one year s worth of triple coincident data , with an angle - averaged inspiral range of equal to , or greater than , 10 mpc for l1 and h1 , and 5 mpc or better for h2 . november 2005 ( l1 started slightly later on 14 november ) until 1 october 2007 , and the performance of the detectors during it is summarised in . one year of triple coincidence was achieved on 21 september 2007 , with a total triple coincidence duty factor of 52.5% for the whole run . the average insprial range over s5 was 15 mpc for h1 and l1 , and 8 mpc for h2 . after the end of s5 the ligo h2 detector and geo600 were kept operational while possible in an evening and weekend mode called astrowatch . this observing mode continued until early 2009 , after which h2 was retired . during this time commissioning of some upgrades to the 4 km ligo detectors took place for the sixth and final initial ligo science run ( s6 ) the aim of these upgrades , called enhanced ligo , was to try and increase sensitivity by a factor of two . enhanced ligo involved the direct implementation of technologies and techniques designed for the later upgrade to advanced ligo ( see section 6.3.1 ) such as , most notably , higher - powered lasers , a dc readout scheme ( see section 5.4 ) , the addition of output mode cleaners and the movement of some hardware into the vacuum system . the lasers , supplied by the albert einstein institute and manufactured by laser zentrum hannover , give a maximum power of 30 w , which is around 3 times the initial ligo power . the upgrade to higher power required that several of the optical components needed to be replaced . these upgrades were only carried out on the 4 km h1 and l1 detectors due to the h2 detector being left in astrowatch mode during the commissioning period . the upgrades were able to produce 1.52 times sensitivity increases at frequencies above 200 hz , but generally at lower frequencies various sources of noise meant sensitivity increases were not possible . s6 took place from july 2009 until 20 october 2010 , at which point decommissioning started for the full upgrade to advanced ligo . typically the detectors ran with laser power at 10 w during the day ( at higher power the detector was less stable and the higher level of anthropogenic noise during the day meant that achieving and maintaining lock required lower power ) and 20 w at night , leading to inspiral ranges from 1020 mpc . geo600 achieved first lock as a power - recycled michelson ( with no signal recycling ) in late 2001 . commissioning over the following year , detailed in , included increases in the laser power , installation of monolithic suspensions for the end test masses ( although not for the beam splitter and inboard mirrors ) , rearrangement of the optical bench to reduce scattered light and implementation of an automatic alignment system . for the s1 run , carried out in coincidence with ligo ( and , in part , tama300 ) , the detector was kept in this configuration ( see for the status of the detector during s1 ) . it had a very high duty factor of 98% , although its strain sensitivity was 2 orders of magnitude lower than the ligo instruments . the auto - alignment system in geo600 has since meant that it has been able to operate for long periods without manual intervention to regain lock , as has been the case for initial ligo . following s1 the signal recycling mirror was installed and in late 2003 the first lock of the fully dual - recycled system was achieved ( see [ 289 , 318 , 163 ] for information on the commissioning of geo600 as a dual - recycled detector ) . other upgrades included the installation of the final mirrors , suspended as triple pendulums , and with monolithic final stages . once installed it was found that there was a radius of curvature mismatch with one of the mirrors , which had to be compensated for by carefully heating the mirror . due to this commissioning effort geo600 did not participate in the s2 run . very soon after the implementation of dual - recycling geo600 took part in the s3 run . this occurred over two time intervals from 511 november 2003 , dubbed s3i , and from 30 december 2003 to 13 january 2004 , dubbed s3ii . during s3i geo600 operated with the signal - recycling cavity tuned to 1.3 khz , and had a 95% duty factor , but was then taken off - line for more commissioning work . in the period between s3i and ii various sources of noise and lock loss were diagnosed and mitigated , including noise from a servo in the signal recycling cavity and electronic noise on a photo - diode . this lead to improved sensitivity by up to an order of magnitude at some frequencies ( see figure 16 ) . for s3ii the signal recycling cavity was tuned to 1 khz and , due to the upgrades , had an increased duty factor of 99% . geo600 operated during the whole of s4 ( 22 february to 24 march 2004 ) , in coincidence with ligo , with a 97% duty factor . it used the same optical configuration as s3 , but had sensitivity improvements from a few times to up to an order of magnitude over the s3 values . also shown is the theoretical noise budget for the detector when tuned to 550 hz the operating position for the s5 run . the typical strain sensitivities from the geo600 science runs s1 through s5 . also shown is the theoretical noise budget for the detector when tuned to 550 hz the operating position for the s5 run . the main changes to the detector after s4 were to shift the resonance condition of the signal recycling cavity to a lower frequency , 350 hz , allowing better sensitivity in the few hundred hz regime , and increasing the circulating laser power , with an input power of 10 w. the pre - s5 peak sensitivity was 4 10 hz at around 400 hz , with an inspiral range of 0.6 mpc . geo600 did not join s5 at the start of the ligo run , but from 21 january 2006 was in a night - and - weekend data - taking mode whilst noise hunting studies and commissioning were conducted . it went into full - time data taking from 1 may to 16 october 2006 , with an instrumental duty factor of 94% . the average peak sensitivity during s5 was better than 3 10 hz ( see for a summary of geo600 during s5 ) . after this it was deemed more valuable for geo600 to continue more noise hunting and commissioning work , to give as good a sensitivity as possible for when the ligo detectors went offline for upgrading . geo600 continued operating in astrowatch mode between november 2007 and july 2009 after which upgrades began . the plans for the geo600 detector are to continue to use it as a test - bed for more novel interferometric techniques whilst focusing on increasing in sensitivity at higher frequencies ( greater than a few hundred hz ) . the main upgrades started during 2009 were to change the read - out scheme from an rf read - out to a dc read - out system ( also see section 5.4 ) , install an output mode cleaner , place the read - out system in vacuum , injecting squeezed light [ 302 , 114 ] into the output port , and finally increasing the input laser power to 35 w. running the interferometer with squeezed light will be the first demonstration of a full - scale gravitational - wave detector operating beyond the standard quantum limit . geo - hf participated in s6 in an overnight and weekend mode , alongside a commissioning schedule , and is continuing in this mode following the end of s6 . in summer 2002 virgo completed the commissioning of the central area interferometer , consisting of a power - recycled michelson interferometer , but without the 3 km fabry - prot arm cavities . over the next couple of years various steps were made towards commissioning the full - size interferometer . in early 2004 first lock with the 3 km arms was achieved , but without power - recycling , and by the end of 2004 lock with power recycling was achieved . during summer 2005 the commissioning runs provided order - of - magnitude sensitivity improvements , with a peak sensitivity of 6 10 hz at 300 hz , and an inspiral range of over 1 mpc . in late 2005 see [ 58 , 59 , 60 , 61 ] for more detailed information on the commissioning of the detector . virgo joined coincident observations with the ligo and geo600 s5 run with 10 weekend science runs ( wsrs ) starting in late 2006 until march 2007 . over this time improvements were made mainly in the mid - to - low frequency regime ( 300 hz ) . full - time data taking , under the title of virgo science run 1 ( vsr1 ) , began on 18 may 2007 and ended with the end of s5 on 1 october 2007 . during vsr1 , the science - mode duty factor was 81% and by the end of the run maximum neutron - star - binary inspiral range was frequently up to about 4.5 mpc . the best sensitivity curves for wsr1 , wsr10 and vsr1 can be seen in figure 17 . figure 17the best strain sensitivities from the virgo weekend and full time science runs wsr1 , wsr10 , vsr1 and vsr2 [ 305 , 57 ] . the best strain sensitivities from the virgo weekend and full time science runs wsr1 , wsr10 , vsr1 and vsr2 [ 305 , 57 ] . at the same time as commissioning for enhanced ligo was taking place the main upgrade was to the lasers to increase their power from 10 to 25 w at the input mode cleaner , with upgrades also to the thermal compensation system on the mirrors , the control electronics , mode cleaners and injection optics [ 64 , 141 ] . virgo+ started taking data with enhanced ligo for virgo science run 2 ( vsr2 ) and sensitivities of virgo+ close to the initial virgo design sensitivity were reached . this was followed by vsr3 , which began on 11 august 2010 and ran until 20 october 2010 . prior to the advent of the large scale interferometric detectors there had been some limited effort to produce astrophysical results with the prototype interferometers . the caltech 40 m detector was used to search for , and set an upper limit on , the gravitational wave emission from pulsar psr j1939 + 2134 , and on the rate of neutron star binary inspirals in our galaxy , using coincident observations with the university of glasgow prototype and , more recently , on its own . coincident observations using the prototype detectors at the university of glasgow and max planck institute for quantum optics , in garching , germany , were used to set an upper limit on the strain of gravitational wave bursts . the garching detector was used to search for periodic signals from pulsars , and in particular set a limit on a potential source in sn 1987a . however since the start of science data taking for the large scale detectors there has been a rapid rise in the number , and scope , of science result papers being published . with the vastly improved sensitivities pushing upper limits on source populations and strengths towards astrophysically interesting areas . the recent analysis efforts have generally been split into four broad areas depending on the expected signal type : unmodelled transients or bursts , e.g. , supernovae ; modelled transients , e.g. , inspirals and ring - downs ( or more specifically compact binary coalescences , cbc ) ; continuous sources ; stochastic sources . within each area a variety of different sources could exist and a variety of analysis techniques have been developed to search for them . some electromagnetic sources , such as radio pulsars and -ray bursts , are also used to enhance searches . a good review of the data analysis methods used in current searches , and the astrophysical consequences of some of the results described below , can be found in . here we will mainly focus on those produced by the ligo scientific collaboration detectors ligo and geo600 from s1 to the s5 run . at the time of writing not all of the data from the s6 run had been fully analysed , with more results expected over the next year . reviews of some early s5 , and prior science run , results can also be found in [ 254 , 139 ] . in none of the searches searches for unmodelled bursts , e.g. , from supernova core - collapse , are based on looking for short duration periods of excess power in the detectors . transients are common features in the data , so to veto these events from true gravitational - wave signals they must be coincident in time , and to some extent amplitude and waveform , between multiple detectors . various methods to assess instrumental excess power , and inter - detector correlations , are used , some examples of which can be found here [ 202 , 77 , 284 , 228 , 107 , 112 , 113 ] . these algorithms will produce triggers , which are periods of excess power that cross a predetermined signal - to - noise ratio threshold ( determined by tuning the algorithms on a section of playground data , so that the output produces a desired false alarm rate ) . real signals can not be turned off , and detectors can not be shielded from them , so the background rate has to be approximated by time shifting one detector s data stream with respect the the others . time shifts should only leave triggers due to random coincidences in detector noise and there should be no contribution from real signals . once a background is calculated , the statistical significance of the foreground rate can be compared to it . to assess the sensitivity of these searches , hardware ( the interferometer mirrors are physically moved via the control system ) and software signals are injected into the data stream at various strengths and the efficiency of the algorithms at detecting them data from the ligo s1 run was searched for gravitational - wave bursts of between 4 to 100 ms , and within the frequency band 150 to 3000 hz . no plausible candidate event was found , but a 90% confidence upper limit on the event rate of 1.6 events per day was set . the search was typically sensitive , at a 50% detection efficiency , to bursts with amplitudes of hrss 10 10 hz ( defined in terms of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${h_{{\rm{rss } } } } \equiv \sqrt { \int { |h{|^2}{\rm{d}}t}}$\end{document } , which is the root - sum - squared strain amplitude spectral density ) . due , in part , to its lower sensitivity geo600 data was not used in this analysis . the s2 data s improved sensitivity , and advances in the analysis techniques , allowed a sensitivity to signals ( in the frequency range 1001100 hz ) in the amplitude range hrss 10 10 hz . interpreting the best sensitivities astrophysically gave order of magnitude estimates on the visible range of 100 pc for a class of theoretical supernova waveforms , and 1 mpc for the merger of 50 m black holes . again no signal was seen , but a 90% upper limit of 0.26 events per day was set for strong bursts . in the frequency range 7002000 hz tama300 data was also used in the search giving amplitude sensitivities of hrss 13 10 hz and decreasing the rate upper limit to 0.12 events per day . one used the 8 days of triple coincidence data from the three ligo detectors to search for sub - second bursts in the frequency range 1001100 hz . the search was sensitive to signals with amplitudes over hrss 1 10 hz , but did not include an astrophysical interpretation of the limit or event rate upper limit . this run included coincident operation with the italian auriga bar detector and this data has been analysed . this search looked for short bursts of less than 20 ms within the 850950 hz band ( around the bar s sensitive resonant frequency ) . this had comparable sensitivity to the ligo - only s2 search and produced a 90% confidence rate upper limit of 0.52 events / day . for s4 15.5 days of ligo data this was sensitive to signals with hrss 10 and set a 90% confidence rate upper limit of 0.15 per day . these show that there would be a 50% detection efficiency to signals of sine - gaussian nature ( at the most sensitive frequency of 153 hz and quality factor q = 8.9 ) at a distance of 10 kpc for an energy of 10 mc , and would be sensitive to signals out to the virgo cluster ( 16 mpc ) for an energy release of 0.25 mc . there was also a burst search combining s4 geo600 and ligo data for the first time . this searched data between 7682048 hz where the sensitivities were most comparable and used 257 hours of quadruple coincidence between the detectors and saw no gravitational wave events . for the analysis on the first year of s5 data the frequency range for the all - sky burst search was split a low frequency search covered the most sensitive region between 602000 hz , and a high frequency search covering 16 khz ( this being the first time an untriggered burst search looked at frequencies above 3 khz ) . the high frequency search set a 90% upper limit on the rate of 5.4 events per year for strong events . the low frequency search analysed more data than the high frequency and set an event rate limit of 3.6 events per year . the second year of s5 ligo data was analysed with geo600 and virgo vsr1 data to search for bursts over the whole 506000 hz band . combining this with the earlier s5 searches gave hrss upper limits for a variety of simulated waveforms of 6 10 hz to 2 10 hz , and a 90% confidence event rate for signals between 642048 hz of less than two per year . modeled bursts generally mean the inspiral and coalescence stage of binaries consisting of compact objects , e.g. , neutron stars and black holes . the signals are generally well approximated by post - newtonian expansions of the einstein equation , which give the amplitude and phase evolution of the orbit . more recently signal models have started to include numerical relativity simulations of the merger stage . as mentioned in section 6 the best estimate of the number of signals observable with initial ligo at design sensitivity ( i.e. during s5 ) would be 0.02 per year ( based on an event rate of 1 10 per year per mweg ) . the majority of inspiral searches make use of matched filtering in which a template bank of signal models is built [ 250 , 252 ] , with a maximum mismatch between templates that is generally of order 10% . these templates are then cross - correlated with the data and statistically significant triggers ( i.e. times when the template and data are highly correlated ) from this are looked for . triggers must be coincident between detectors and the significance of any trigger is judged against a background calculated in the same way as described in section 6.2.1 . see for a good description of the search method . the first search for an inspiral signal with data from the ligo s1 run looked for compact object coalescences with component masses between 13 m and was sensitive to such sources within the milky way and magellanic clouds . it gave a 90% confidence level upper limit on the rate of 170 per year per mweg . for the s2 ligo analysis the search was split into 3 areas covering neutron - star binaries , black - hole binaries and primordial black - hole binaries in the galactic halo . the neutron - starbinary search used 15 days of data with coincidence between either h1 and l1 or h2 and l1 . it had a range of 1.5 mpc , which spanned the local group of galaxies , and gave a 90% event rate upper limit on systems with component masses of 13 m of 47 per year per mweg . the black - hole - binary search looked for systems with component masses in the 320 m range using the same data set as the neutron - star - binary search . this search had a 90% detection efficiency for sources out to 1 mpc and set a 90% rate upper limit of 38 per year per mweg . the third search looked for low mass ( 0.21 m ) primordial black - hole binaries in a 50 kpc radius halo surrounding the milky way . this placed a 90% confidence - rate upper limit of 63 events per year per milky way halo . the s2 search was performed in coincidence with the tama300 dt8 period and an inspiral search for neutron - star binaries was performed on data when tama300 and at least one of the ligo sites was operational . this gave a total of 584 hours of data for the analysis , which set a 90% rate upper limit of 49 per year per mweg , although this search was only sensitive to sources within the majority of the milky way . the search for neutron - star - black - hole binaries in s3 ligo data used techniques designed specifically for systems with spinning components . it searched for systems with component masses in the range 120 m and analysed 167 hours of triple coincident data and 548 hours of h1h2 data to set the upper limits . for a typical system with neutron - star and black - hole mass distributions centred on 1.35 m and 5 m ( from the population statistics discussed in ) this search produced a 90% confidence - rate upper limit of 15.9 per year per l10 . the search for a wide range of binary systems with components consisting of primordial black holes , neutron stars , and black holes with masses in the ranges given above 788 hours of s3 data and 576 hours of s4 data were used and no plausible gravitational - wave candidate was found . the highest mass range for the black - holebinary search was set at 40 m for s3 and 80 m for s4 . at peak in the mass distribution of these sources 90% confidence - rate upper limits were set at 4.9 per year per l10 for primordial black holes , 1.2 per year per l10 for neutron - star binaries , and 0.5 per year per l10 for black - hole - binaries . s4 data has also been used to search for ring - downs from perturbed black holes , for example following black - hole - binary coalescence . the search was sensitive to ring - downs from 10500 m black holes out to a maximum range of 300 mpc , and produced a best 90% confidence upper limit on the rate of ring - downs to be 1.6 10 per year per l10 for the mass range 85390 m. one other kind of modeled burst search is that looking for gravitational waves produced by cusps in cosmic ( super)strings . just over two weeks of ligo s4 data were used to search for such signals . this was used to constrain the rate and parameter space ( string tension , reconnection probability , and loop sizes ) , but was not able to beat limits set by big bang nucleosynthesis . data from the first and second year of s5 ( prior to virgo joining with vsr1 ) have been searched for low - mass binary coalescences with total masses in the range 235 m. the second year search results have produced the more stringent upper limits with 90% confidence rates for neutron - star - binaries , black - hole - binaries and neutron - star - black - hole systems respectively of 1.4 10 , 7.3 10 and 3.6 10 per year per l10 . five months of overlapping s5 and vsr1 data were also searched for the same range of signals giving 90% confidence upper rates of 8.7 10 per year per l10 , 2.2 10 per year per l10 , and 4.4 10 per year per l10 . the whole 2 years of ligo s5 data were also used to search for higher mass binary coalescences with component mass between 199 m and total masses of 25100 m. no signal was seen , but a 90% confidence upper limit rate on mergers of black - hole - binary systems with component masses between 19 and 28 m , and with negligible spin , was set at 2.0 mpc myr . many gravitational wave burst sources will be associated with electromagnetic ( or neutrino ) counterparts , for example short -ray bursts ( grbs ) are potentially caused by black - hole and neutronstar coalescences . joint observation of a source as both a gravitational wave and electromagnetic event also greatly increases the confidence in a detection . therefore many searches have been performed to look for bursts coincident ( temporally and spatially ) with external electromagnetic triggers , such as grbs observed by swift for example . these searches have used both excess power and modeled matched - filter methods to look for signals . during s2 a particularly bright -ray burst event ( grb 030329 ) occurred and was specifically targeted using data from h1 and h2 . the search looked for signals with duration less than 150 ms and in the frequency range 802048 hz . this produced a best strain upper limit for an unpolarised signal around the most sensitive region at 250 hz of hrss = 6 10 hz . the first target was the hyperflare from the soft -ray repeater sgr 180620 ( sgrs are thought to be magnetars , neutron stars with extremely large magnetic fields of order 10 gauss ) on 27 december 2004 ( this actually occurred before s4 in a period when only the h1 detector was operating ) . the search looked for signals at frequencies corresponding to short duration quasi - periodic oscillations ( qpos ) observed in the x - ray light curve following the flare . the most sensitive 90% upper limit was for the 92.5 hz qpo at hrss = 4.5 10 hz , which corresponds to an energy emission limit of 4.3 10 mc ( of the same order as the total electromagnetic emission assuming isotropy ) . the other search used ligo data from s2 , s3 and s4 to look for signals associated with 39 short duration -ray bursts ( grbs ) that occurred in coincidence with these runs . the grb triggers were provided by ipn , konus - wind , hete-2 , integral and swift as distributed by the grb coordinate network . the search looked in a 180-second window around the burst peak time ( 120 seconds before and 60 seconds after ) and for each burst there were at least two detectors contributing data . no signal coincident with a grb was observed and the sensitivities were not enough to give any meaningful astrophysical constraints , although simulations suggest that for s4 , as in the general burst search , it would have been sensitive to sine - gaussian signals out to tens of mpc for an energy release of order a solar mass . the first search of virgo data in coincidence with a grb was performed on data from a commissioning run in september 2005 . the long duration grb 050915a was observed by swift on 15 september 2005 and virgo data was used to search for an unmodelled burst in a window of 180 seconds around ( 120 s before and 60 s after ) the grb peak time . the search produced a strain upper limit of order 10 in the frequency range 2001500 hz , but was mainly used as a test - bed for setting up the methodology for future searches , including coincidence analysis with ligo . data from the s5 run has been used to search for signals associated with even more -ray bursts . one search looked specifically for emissions from grb 070201 [ 156 , 155 ] , which showed evidence of originating in the nearby andromeda galaxy ( m31 ) . the data around the time of this burst was used to look for an unmodelled burst and an inspiral signal as might be expected from a short grb . the analysis saw no gravitational - wave event associated with the grb , but ruled out the event being a neutron - star - binary inspiral located in m31 with a 99% confidence . again , assuming a neutron - star - binary inspiral , but located outside m31 , the analysis set a 90% confidence limit that the source must be at a distance greater than 3.5 mpc . assuming a signal again located in m31 , the unmodelled burst search set an upper limit on the energy emitted via gravitational waves of 4.4 10 mc , which was well within the allowable range for this being an sgr hyper - flare in m31 . searches for 137 grbs ( both short and long grbs ) that were observed , mainly with the swift satellite , during s5 and vsr1 have been performed again using unmodelled burst methods and for ( 22 short bursts ) inspiral signals . the unmodeled burst observations were used to set lower limits on the distance to each grb , with typical limits , assuming isotropic emission , at \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$d \sim 15{\mkern 1mu } { \text{mpc}}{(e_{{\text{gw}}}^{{\text{iso}}}/0.01{\mkern 1mu } { m _ \odot } { c^2})^{1/2}}$\end{document}. the inspiral search , which was sensitive to cbcs with total system masses between 2 m and 40 m , was able to exclude with 90% confidence any bursts being neutron - star - black - hole mergers within 6.7 mpc , although the peak distance distribution of grbs is well beyond this . another search has been to look for gravitational waves associated with flares from known sgrs and anomalous x - ray pulsars ( axps ) , both of which are thought to be magnetars . during the first year of s5 there were 191 ( including the december 2004 sgr 180620 event ) observed flares from sgrs 180620 and 1900 + 14 for which at least one ligo detector was online , and 1279 flare events if extending that to six known galactic magnetars and including all s5 and post - s5 astrowatch data including virgo and geo600 . the data around each event was searched for ring - down signals in the frequency range 13 khz and with decay times 100400 ms as might be expected from f - mode oscillations in a neutron star . no gravitational bursts were seen from any of the events . for the earlier search the lowest 90% upper limit on the gravitational - wave energy from the ring - down search was \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$e_{{\rm{gw}}}^{90\% } = 2.4 \times { 10^{48}}$\end{document } erg for an sgr 180620 burst on 24 august 2006 . the lowest 90% upper limit on the unmodeled search was \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$e_{{\rm{gw}}}^{90\% } = 2.9 \times { 10^{45}}$\end{document } erg for an sgr 180620 burst on 21 july 2006 . the smallest limits on the ratio of energy emitted via gravitational waves to that emitted in the electromagnetic spectrum were of order 10100 , which are into a theoretically - allowed range . the latter search gave the lowest gravitational - wave emission - energy upper limits for white noise bursts in the detector - sensitive band , and for f - mode ring - downs ( at 1090 hz ) , of 3.0 10 erg and 1.4 10 erg respectively , assuming a distance of 1 kpc . the f - mode energy limits approach the range seen emitted electromagnetically during giant flares . one of these flares , on 29 march 2006 , was actually a storm of many flares from sgr 1900 + 14 . for this event a more sensitive search has been performed by stacking data around the time of each flare . waveform dependent upper limits of the gravitational - wave energy emitted were set between 2 10 erg and 6 10 erg , which are an order of magnitude lower than the previous upper limit for this storm ( included in the search of ) and overlap with the range of electromagnetic energies emitted in sgr giant flares . another possible source of gravitational waves associated with electromagnetically - observed phenomenon are pulsar glitches . during these it is possible that various gravitational - wave - emitting vibrational modes of the pulsar may be excited . a search has been performed for fundamental modes ( f - modes ) in s5 data following a glitch observed in the timing of the vela pulsar in august 2006 . over the search frequency range of 13 khz this provided upper limits on the peak strain of 0.61.4 10 depending on the spherical harmonic that was excited . already efforts are under way to invert this process of searching gravitational - wave data for external triggers , and instead supplying gravitational - wave burst triggers for electromagnetic follow - up . this is being investigated across the range of the electromagnetic spectrum from radio , through optical ( e.g. , [ 198 , 119 ] ) and x - ray/-ray , and even looking for coincidence with neutrino detectors [ 86 , 258 , 111 ] . having multi - messenger observations can have a large impact on the amount of astrophysical information that can be learnt about an event . there are fully targeted searches , which look for gravitational waves from known radio pulsars in which the position and spin evolution of the objects are precisely known . there are semi - targeted searches , which look at potential sources in which some , but not all , the source signal parameters are known , for example neutron stars in x - ray binary systems , or sources in supernova remnants where no pulses are seen , which have known position , but unknown frequency . finally , there are all - sky broadband searches in which none of the signal parameters are known . the targeted searches tend to be most sensitive as they are able to perform coherent integration over long stretches of data with relatively low computational overheads , and have a much smaller parameter space leading to fewer statistical outliers . due to various neutron - star population statistics , creation rates and energetics arguments , there is an estimate that the amplitude of the strongest gravitational - wave pulsar observed at earth will be h0 4 10 ( a more thorough discussion of this argument can be found in ) , although this does not rule out stronger sources . the various search techniques used to produce these results all look for statistically - significant excess power in narrow frequency bins that have been doppler demodulated to take into account the signal s shifting frequency caused by the earth s orbital motion with respect to the source ( or also including the modulations to the signal caused by the source s own motion relative to the earth , such as for a pulsar in a binary system ) . the statistical significance of a measured level of excess power is compared to what would be expected from data that consisted of gaussian noise alone . a selection of the searches are summarised in , but for more detailed descriptions of the various methods see [ 100 , 206 , 188 , 33 , 29 , 132 ] . in s1 a fully - coherent targeted search for gravitational waves from the then - fastest millisecond pulsar j1939 + 2134 was performed . this analysis and the subsequent lsc known - pulsar searches assume that the star is triaxial and emitting gravitational waves at exactly twice its rotation frequency . all the data from ligo and geo600 was analysed and no evidence of a signal was seen . a 95% degree - of - belief upper limit on the gravitational - wave strain amplitude was set using data from the most sensitive detector , l1 , giving a value of 1.4 10 . this result was also interpreted as an ellipticity of the star given a canonical moment of inertia of 10 kg m at = 2.9 10 . however , this was still of order 100 000 times higher than the limit that can be set by equating the star s rate of loss of rotational kinetic energy with that emitted via gravitational radiation the number of known pulsar sources searched for with ligo data increased from 1 to 28 , although all of these were isolated pulsars ( i.e. not in binary systems , although potentially still associated with supernova remnants or globular clusters ) . this search used pulsar timing data supplied by lyne and kramer from jodrell bank observatory to precisely reconstruct the phase of the gravitational - wave signal over the period of the run . the lowest 95% upper limit on gravitational - waves amplitude was 1.7 10 for psr j19105959d , and the smallest upper limit on ellipticity ( again assuming the canonical moment of inertia ) was 4.5 10 for the relatively - close pulsar psr j21243358 , at a distance of 0.25 kpc . the pulsar closest to its inferred spin - down limit was the crab pulsar ( psr j0534 + 2200 ) with an upper limit 30 times greater than that from spin - down . s2 also saw the use of two different all - sky - wide frequency band searches that focused on isolated sources , but also including a search for gravitational waves from the low mass x - ray binary scorpius x1 ( sco - x1 ) . the first search used a semi - coherent technique to search 60 days of s2 data in the frequency band between 200400 hz and with signal spin - downs between 1.1 10 and 0 hz s . this gave a lowest gravitational - wave strain 95% upper limit of 4.4 10 for the l1 detector at around 200 hz . the other all - sky search was fully coherent and as such was computationally limited to only use a few hours of the most sensitive s2 data . it searched frequencies between 160728.8 hz and spin - downs less than 4 10 hz s for isolated sources and gave a 95% upper limit across this band from 6.6 10 to 1 10 . the search for gravitational waves from sco - x1 used the same period of data . it did not have to search over sky position as this is well known , but did have to search over two binary orbital parameters the projected semi - major axis and the orbital phase reference time . the frequency ranges of this search relied on estimates of the spin - frequency from quasi - periodic oscillations in the x - rays from the source and covered two 20 hz bands from 464484 hz and 604624 hz ( it should be noted that it is now thought that these estimates of the spin - frequency are unreliable ) . in these two ranges one search that was carried out purely on ligo s3 data was the coherent all - sky wide - band isolated pulsar search using the distributed computing project einstein@home . the project is built upon the berkeley open infrastructure for network computing and allows the computational workload to be distributed among many computers generally contributed by the general public who sign up to the project . this used the most sensitive 600 hours of data from h1 and cut it into 60 ten hour stretches on each of which a coherent search could be performed . the data was farmed out to computers owned by participants in the project and ran as a background process or screen saver . the search saw no plausible gravitational - wave candidates and the result is described at , but it was not used to produce an upper limit . in the known pulsar search the number of sources searched for using the combined ligo data from s3 and s4 was increased to 78 . for many of the pulsars that overlapped with the previous s2 analysis results were improved by about an order of magnitude . the lowest 95% upper limit on gravitational - waves amplitude was 2.6 10 for psr j16037202 , and the smallest ellipticity was again for psr j21243358 at just less than 10 . the upper limit for the crab pulsar was found to be only 2.2 times above that from the spin - down limit . three different , but related , semi - coherent all - sky continuous wave searches were performed on s4 ligo data , looking for isolated neutron stars in the frequency range from 501000 hz and the spin - down range from 1 10 to 0 hz s . the best 95% upper limit based on an isotropically - distributed , randomly - oriented for one of the searches , which combined data from the different detectors , an isolated pulsar emitting at near 100 hz , and with an extreme ellipticity of 10 could have been seen at a distance of 1 kpc , although for a more realistic ellipticity of 10 only a distance of less than 1 pc would be visible over the entire ligo band . the einstein@home project was also used to search the most sensitive data from s4 , which consisted of 300 hours of h1 data and 210 hours of l1 data . the search performed a coherent analysis on 30-hour stretches of this data and covered the frequency range of 501500 hz . the range of spin - downs was chosen by using a minimum spin - down age and having f/ < < 0.1 f/ ( small spin - ups are allowed as some pulsars in globular clusters exhibit this due to their doppler motions within the clusters ) , with = 1000 years for signals below 300 hz and = 10 000 years above 300 hz . approximately 6000 years of computational time spread over about 100 000 computers were required to perform the analysis . no plausible gravitational - wave candidates were found , although the results suggest that 90% of sources with strain amplitudes greater than 10 would have been detected by the search . a search designed to produce a sky map of the stochastic background was also used to search for gravitational waves from sco - x1 using a method of cross - correlating h1 and l1 data . this produced a 90% root - mean - squared upper limit on gravitational wave strain of h = 3.4 10(f/200 hz ) for frequencies greater than 200 hz . the first 8 months of s5 have been used to perform an all - sky search for periodic gravitational waves . this search used a semi - coherent method to look in the frequency range 501100 hz and spin - down range 5 10 0 hz s and used data from the h1 and l1 detectors . it obtained 95% strain upper limits of less than 10 over a frequency band of 200 hz . the search would have been sensitive to a neutron star with equatorial ellipticity greater than 10 within around 500 pc . einstein@home has been used to search for periodic waves of 501500 hz in 860 hours of data from a total span of 66 days of s5 data . it would have been sensitive to 90% of sources in the 125225 hz band with amplitudes greater than 3 10 . the first approximately 9 months of s5 data was used for a coherent search for gravitational waves from the crab pulsar . in this search two methods were used : the first followed the method of the targeted search and assumed that the gravitational waves are phase locked to the electromagnetic pulses ; the second allowed for some mechanism , which would cause a small mismatch between the two phases . two 95% upper limits were set , one using astrophysical constraints on the pulsar orientation angle and polarisation angle and the other applying no such constraints . with the first method these 95% upper limits were 3.4 10 and 2.7 10 respectively , which correspond to ellipticities of 1.8 10 and 1.4 10 ( assuming the canonical moment of inertia ) . these beat the crab pulsar s spin - down limit by 4 to 5 times and can be translated into the amount of the available spin - down power that is emitted via gravitational waves , with the lower of these limits showing that less than 4% of power is going into gravitational waves . for the second search the uniform and restricted prior analyses gave upper limits of 1.7 10 and 1.2 10 respectively . the whole of s5 was used to search for emissions from 116 known pulsars . during this search the crab limit was further brought down to be less than a factor of 7 below the spin - down limit , and the spin - down limit is reached for one other pulsar psr j05376910 . of the other pulsars , the best ( lowest ) upper limit on gravitational - wave amplitude was 2.3 10 for psr j16037202 and our best ( lowest ) limit on the inferred pulsar ellipticity is 7.0 10 for psr j21243358 . a semi - targeted search was performed with 12 days of s5 data , although this time searching for a source with a known position in the cassiopeia a ( cas a ) supernova remnant , but for which there is no known frequency . the search looked in the frequency band between 100300 hz and covered a wide range of first and second frequency derivatives and no signal was seen , but it gave 95% amplitude and ellipticity upper limits over the band of ( 0.71.2 ) 10 and ( 0.44 ) 10 respectively . these results beat indirect limits on the emission based on energy - conservation arguments ( similar , but not the same as the spin - down limits ) and were also the first results to be cast as limits on the r - mode amplitude . the vela pulsar has a spin frequency of 11 hz and was not accessible with current ligo data . however , virgo vsr2 data had sensitivity in the low frequency band that made a search for it worthwhile . using 150 days of virgo data , no signal was seen , but a 95% upper limit on the amplitude of 2 10 was set , which beat the spin - down limit by 1.6 times . other than the crab pulsar , this is currently the only other object for which the spin - down limit has been beaten . searches are conducted for a cosmological , or astrophysical , background of gravitational waves that would show up as a coherent stochastic noise source between detectors . this is done by performing a cross - correlation of data from two detectors as described in . in s1 the most sensitive detector pair for this correlation was h2-l1 ( the h1h2 pair are significantly hampered by local environmental correlations ) and they gave a 90% confidence upper limit of gw < 44 9 within the 40314 hz band , where the upper limit is in units of closure density of the universe and for a hubble constant in units of 72 km s mpc . this limit was several times better than previous direct - detector limits , but still well above the concordance acdm cosmology value of the total energy density of the universe of 0 1 ( see , e.g. , ) . no published stochastic background search was performed on s2 data , but s3 data was searched and gave an upper limit that improved on the s1 result by a factor of 10 . the most sensitive detector pair for this search was h1-l1 for which 218 hours of data were used . upper limits were set for three different power - law spectra of the gravitational - wave background . for a flat spectra , as predicted by some inflationary and cosmic string models , a 90% confidence upper limit of gw(f ) = 8.4 10 in the 69156 hz range was set ( again for a hubble constant of 72 km s mpc ) . this is still about 60 times greater than a conservative bound on primordial gravitational waves set by big - bang nucleosynthesis ( bbn ) . for a quadratic power law , as predicted for a superposition of rotating neutron - star signals , an upper limit of gw(f ) = 9.4 10(f/100 hz ) was set in the range 73244 hz , and for a cubic power law , from some pre - big - bang cosmology models , an upper limit of gw(f ) = 8.1 10(f/100 hz ) in the range 76329 hz was produced . for s4 354 hours of h1-l1 data and 333 hours of h2-l1 data were used to set a 90% upper limit of gw(f ) < 6.5 10 on the stochastic background between 51150 hz , for a flat spectrum and hubble constant of 72 km s mpc . this result is still several times higher than bbn limits . about 20 days of h1 and l1 s4 data was also used to produce an upper limit map on the gravitational wave background across the sky as would be appropriate if there was an anisotropic background dominated by distinct sources . this search covered a frequency range between 501800 hz and had spectral power limits ( which come from the square of the amplitude h ) ranging from 1.2 10 hz ( 100 hz / f ) and 1.2 10 hz(100 hz / f ) for an f source power spectrum , and limits of 8.5 10 hz and 6.1 10 hz for a flat spectrum . data from s4 was also used to perform the first cross - correlation between an interferometric and bar detector to search for stochastic backgrounds . l1 data and data from the nearby allegro bar detector were used to search in the frequency range 850950 hz , several times higher than the ligo only searches . a 90% upper limit on the closure density of gw(f ) 1.02 ( for the above hubble constant ) was set , which beat previous limits in that frequency range by two orders of magnitude . this limit beats what would be achievable with llo - lho cross correlation of s4 data in this frequency range by a factor of several tens , due to the physical proximity of llo and allegro . the entire two years of s5 data from the ligo detectors has been used to set a limit on the stochastic background around 100 hz to be gw(f ) < 6.9 10 at 95% confidence ( for a flat gravitational - wave spectrum ) . all the current detectors have upgrades planned over the next several years . these upgrades will give rise to the second generation of gravitational - wave detectors , which should start to open up gravitational - wave astronomy as a real observational tool . there are also currently plans being made for third generation detectors , which could provide the premier gravitational - wave observatories for the first half of the century . a brief summary of the planned upgrades to current and future detectors is given below . some of the technologies for these upgrades are discussed earlier in this review ( e.g. section 5 ) . advanced ligo ( aligo ) [ 166 , 214 , 66 ] and advanced virgo ( advvirgo ) [ 62 , 67 ] are the second generation detectors . they are planned to have a sensitivity increase over the levels of the initial detectors by a factor of 1015 times . these increased sensitivity levels would expand the volume of space observed by the detectors by 1000 times meaning that there is a realistic detection rate of neutron - star - binary coalescences of around 40 yr [ 3 , 205 ] . the technological issues required to reach these sensitivities , such as choice of test mass and mirror coating materials , suspension design , interferometric layout , control and readout , would need a separate review article to themselves , but we shall very briefly summarise them here . advanced ligo will consist of three 4 km detectors in the current ligo vacuum system ; two at the hanford site and one at livingston . it will apply some of the technologies from the geo600 interferometer , such as the use of a signal recycling mirror at the output port and monolithic silica suspensions for the test masses , rather than the current steel wire slings . larger test masses will be used with an increase from 11 to 40 kg , although the masses will still be made from fused silica . the mirror coating is likely to consist of multiple alternating layers of silica and tantala , with the tantala layers doped with titania to reduce the coating thermal noise . the seismic isolation systems will be replaced with improved versions offering a seismic cut - off frequency of 10 hz as opposed to the current cut - off of 40 hz . as stated for enhanced ligo ( in section 6.1.2 ) , the laser power will be greater than for initial ligo and a dc readout scheme will be used . initial / enhanced ligo was shut down to begin the installation of these upgrades on 20 october 2010 . the design strain amplitude sensitivity curve for aligo ( and advvirgo and lcgt ) is shown in figure 18 . figure 18design sensitivity curves for the advanced ligo , advanced virgo and lcgt second - generation detectors . the advanced ligo curve comes from , the advanced virgo curve comes from , and the lcgt curve comes from . these curves are based on specific configurations of the detectors and are therefore subject to change . design sensitivity curves for the advanced ligo , advanced virgo and lcgt second - generation detectors . the advanced ligo curve comes from , the advanced virgo curve comes from , and the lcgt curve comes from . these curves are based on specific configurations of the detectors and are therefore subject to change . advvirgo will apply similar upgrades to those for aligo and over a similar timescale ( for details see and ) . plans are to add a signal recycling mirror , monolithic suspensions , increased laser power to 200 w , improved coatings , and to potentially use non - gaussian beams ( see , e.g. , ) , although this option is unlikely . the seismic isolation system will not be changed . the large - scale cryogenic gravitational - wave telescope ( lcgt ) [ 234 , 247 , 207 ] is a planned japanese detector to be sited underground in the kamioka mine . the lgct will consist of a detector with 3 km arms , using sapphire mirrors and sapphire suspensions . initially it will operate at room temperature , but will later be cooled to cryogenic temperatures . this detector is planned to have similar sensitivities to aligo and advvirgo , with a reach for binary coalescences of about 200 mpc with snr of 10 . there currently exists a technology demonstrator called the cryogenic laser interferometer observatory ( clio ) [ 324 , 164 ] , which has a 100 m baseline and is also sited in the kamioka mine . this is to demonstrate the very stable conditions ( i.e. , low levels of seismic noise ) existing in the mine and also the cryogenically - cooled sapphire mirrors suspended from aluminium wires . in experiments with clio at room temperature ( i.e. 300 k ) , using a metallic glass called bolfur for its wire suspensions , it has already been used to produce an astrophysics result by looking for gravitational waves from the vela pulsar , giving a 99.4% confidence upper limit of h = 5.3 10 . tests with the cryogenic system activated and using aluminium suspensions allowed two mirrors to be cooled to 14 k. having a network of comparably - sensitive detectors spread widely across the globe is vital to gain the fullest astrophysical insight into transient sources . position reconstruction for sources relies on triangulating the location based on time - of - flight delays observed between detectors . therefore , having long baselines , and different planes between as many detectors as possible , gives the best positional reconstruction in it is shown that for the 2 us aligo sites sky localisation will be on the order of 1000 square degrees , whereas this can be brought down to a few square degrees with the inclusion of more sites and detectors . observation with multiple detectors also provides the best way to give confidence that a signal is a real gravitational wave rather than the accidental coincidence of background noise . finally , multiple , differently - oriented , detectors will increase the ability to reconstruct a transient sources waveform and polarisation . currently design studies are under way for a third - generation gravitational - wave observatory called the einstein telescope ( et ) . this is a european commission funded study with working groups looking into various aspects of the design including the site location and characteristics ( e.g. underground ) , suspensions technologies ; detector topology and geometry ( e.g. an equilateral triangle configuration ) ; and astrophysical aims . the preliminary plan is to aim for an observatory , which improves upon the second - generation detectors by an order of magnitude over a broad band . there are many technological challenges to be faced in attempting to make this a reality and research is currently under way into a variety of these issues . investigations into the interferometric configuration have already been studied ( see [ 145 , 174 , 178 ] ) , with suggestions including a triple interferometer system made up from an equilateral triangle , an underground location , and potentially a xylophone configuration ( two independent detectors covering different frequency ranges , i.e. , ultimately giving six detectors in total , although constructed over a period of years ) . three potential sensitivity curves are plotted in figure 19 for different configurations of detectors . figure 19potential sensitivities of the einstein telescope for 3 different design concepts : et - b , et - c and et - d . the curves are available from potential sensitivities of the einstein telescope for 3 different design concepts : et - b , et - c and et - d . some of the most interesting gravitational - wave signals , resulting from the mergers of supermassive black holes in the range 10 to 10 m and cosmological stochastic backgrounds , will lie in the frequency region below that of ground - based detectors . the most promising way of looking for such signals is to fly a laser interferometer in space , i.e. to launch a number of drag - free spacecraft into orbit and to compare the distances between test masses in these craft using laser interferometry . until early 2011 , the laser interferometer space antenna ( lisa ) see , for example , [ 125 , 272 , 217 , 216 ] was under consideration as a joint esa / nasa mission as one l - class candidate within the esa cosmic visions program . funding constraints within the us now mean that esa must examine the possibility of flying an l - class mission with european - only funding . accordingly all three l - class candidates are undergoing a rapid redesign phase with the goal of meeting the new european - only cost cap . financial , programmatic and scientific issues will be reassessed following the redesigns and it is currently expected that the selection of the first l - class mission will take place in 2014 . however , for the rest of this article we will discuss the plans for lisa prior to these developments . lisa would consist of an array of three drag - free spacecraft at the vertices of an equilateral triangle of length of side 5 10 km , with the cluster placed in an earth - like orbit at a distance of 1 au from the sun , 20 behind the earth and inclined at 60 to the ecliptic . a current review of lisa technologies , with expanded discussion of , and references for , topics touched upon below , can be found in . here we will focus upon a couple of topics regarding the interferometry needed to give the required sensitivity . proof masses inside the spacecraft ( two in each spacecraft ) form the end points of three separate , but not independent , interferometers . each single two - arm michelson - type interferometer is formed from a vertex ( actually consisting of the proof masses in a central spacecraft ) , and the masses in two remote spacecraft as indicated in figure 21 . the three - interferometer configuration provides redundancy against component failure , gives better detection probability , and allows the determination of the polarisation of the incoming radiation . the spacecraft , which house the optical benches , are essentially there as a way to shield each pair of proof masses from external disturbances ( e.g. , solar radiation pressure ) . drag - free control servos enable the spacecraft to follow the proof masses to a high level of precision , the drag compensation being effected using proportional electric thrusters . illumination of the interferometers is by highly - stabilised laser light from nd : yag lasers at a wavelength of 1.064 microns , laser powers of 2 w being available from monolithic , non - planar ring oscillators , which are diode pumped . for lisa to achieve its design performance strain sensitivity of around 10 hz , adjacent arm lengths have to be sensed to an accuracy of about 10 pm(hz ) . because of the long distances involved and the spatial extent of the laser beams ( the diffraction - limited laser spot size , after travelling 5 10 km , is approximately 50 km in diameter ) , the low photon fluxes make it impossible to use standard mirrors for reflection ; thus , active mirrors with phase locked laser transponders on the spacecraft will be implemented . telescope mirrors will be used to reduce diffraction losses on transmission of the beam and to increase the collecting area for reception of the beam . with the given laser power , and using arguments similar to those already discussed for ground - based detectors with regard to photoelectron shot noise considerations , means that for the required sensitivity the transmitting and receiving telescope mirrors on the spacecraft will have diameters of 40 cm . figure 20a design sensitivity amplitude spectral density curve for lisa created using the standard parameters in the online generator at . the curve assumes equal length arms , sensitivity averaged over the whole sky and all polarisations , and an snr of 1 . also included is a curve showing the expected background noise from galactic white - dwarf - binary systems , which will dominate over the instrumental noise in the range from 0.11 mhz . a design sensitivity amplitude spectral density curve for lisa created using the standard parameters in the online generator at . the curve assumes equal length arms , sensitivity averaged over the whole sky and all polarisations , and an snr of 1 . also included is a curve showing the expected background noise from galactic white - dwarf - binary systems , which will dominate over the instrumental noise in the range from 0.11 mhz . further , just as in the case of the ground - based detectors , the presence of laser frequency noise is a limiting factor . it leads to an error in the measurement of each arm length . if the arms are equal , these errors cancel out , but if they are unequal , the comparison of lengths used to search for gravitational waves may be dominated by frequency noise . for the 5 10 m long arms of lisa , then , for a relative arm length measurement of 2 10 m hz ( the error budget level allowed in the lisa design for this noise source ) , equation ( 14 ) suggests that a laser stability of 6 10 hz hz is required , a level much better than can be achieved from the laser on its own . the first method of stabilisation is to lock the frequency of one laser in the system on to a local frequency reference , e.g. , a fabry - prot cavity mounted on one of the craft ( see , for example , ) , and then to effectively transfer this stability to other lasers in the system by phase locking techniques . with the temperature fluctuations inside each craft limited in the region of 3 mhz to approximately 10 k hz by three stages of thermal insulation , a cavity formed of material of low expansion coefficient such as ule allows a stability level of approximately 30 hz hz ( again at 3 mhz ) . this level of laser frequency noise is clearly much worse than the required 1.2 10 hz hz ( at 3 mhz ) and a further correction scheme is needed . a second possible stage of frequency stabilisation is arm - locking , which relies on the fact that , by design , the fractional stability of the lisa arms is of order /l 10 hz to derive an error signal from the phase difference between the local laser and the received light . as the received light is phase locked with the local laser from the craft that sent it , it caries a replica of the frequency noise of the local laser noise delayed by one round trip time = 33 s. using this fact , this noise can be suppressed at frequencies smaller than the round trip frequency f = 1/ = 30 mhz . this scheme requires no additional hardware and can be completely implemented in software , but it will still leave frequency noise that is several orders of magnitude above required levels . a third stage frequency stabilisation scheme , which is a post - processing step , is time - delay interferometry ( tdi ) . this makes use of the fact that , because the beams coming down each arm are not combined , the phase of each beam can be measured and recorded . therefore , correlations in the frequency noise can be calculated and subtracted by algebraically combining phase measurements from different craft delayed by the multiples of the time delay between the spacecraft . the accuracy of this is set by the phase measurement accuracy , which allows frequency noise subtraction to below the required level . a simple tdi scheme , for a much simplified constellation , was first based in the frequency domain , but due to complexities in taking into account changing arm lengths and a more complex interferometric scheme , subsequent implementations have been in the time domain . a mathematical overview of the tdi scheme , along with moving spacecraft and unequal arm lengths , can be found in . one of the major components of lisa is the disturbance reduction system ( drs ) , which is responsible for making sure the test masses follow , as far as possible , purely gravitational orbits . this consists of the gravitational reference sensor ( grs ) and the control and propulsion systems used to keep the spacecraft centred on the test mass . the test masses for lisa are 1.96 kg cubes , with sides of 46 mm and made of an alloy of 75% gold and 25% platinum , chosen because of its very small magnetic susceptibility . the masses are housed in a cube of electrodes designed to capacitively sense their position and to have measurement noise levels of 1.8 nm hz . the masses need to be tightly held in place during launch and then released , so a caging mechanism has been designed consisting of 8 hydraulic fingers ( one for each corner of the mass ) pushing with 1200 n of force . there will be adhesion between the fingers and the masses , which will require about 10 n of force per finger to break . to provide this force two plungers will push on the the top and bottom surfaces of the masses releasing them from the fingers , followed by pushing smaller release tips in each plunger , and quickly retracting them , to overcome their adhesion to the masses . charged particles produced by cosmic radiation interacting with the surrounding spacecraft can cause the test masses to become charged at a rate of about 50 electrons per second . current plans are to use uv light from mercury lamps ( or potentially uv leds ) to discharge the masses . another key technology for the drs are the micro - newton thrusters , which provide the fine control needed for drag - free flight . these will mainly be used to counteract solar radiation pressure on the spacecraft , which requires about 10 n per relevant thruster . thrust noise as a function of frequency is required to be smaller than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$0.1\mu { \rm{n}}\;{\rm{h}}{{\rm{z}}^{{\rm{- 1/2 } } } } \times \sqrt { 1 + { { \left({{{10\;{\rm{mhz } } } \over f } } \right)}^4}.}$$\end{document } two types of system , both of which meet the requirements , will be tested on lisa pathfinder : the us colloid micro - newton thruster ( cmnt ) ; and the european field emission electric propulsion system ( feep ) . the cmnt uses small drops of a colloid , which it ionises through field emission , accelerates and ejects from the thruster . two designs of feep currently exist , one using indium and the other caesium and with different geometries , which , instead of ionising a droplet of colloid , just use single ions . many of the systems above are being tested in the lisa pathfinder mission ( see below ) and use the nominal lisa designs . there are many other issues associated with laser interferometry , and other aspects of the mission mentioned above , for lisa , which are not dealt with here and the interested reader should refer to [ 183 , 192 , 193 ] for a discussion of some of these . for lisa an industrial contract was awarded to astrium gmbh for the lisa mission formulation study . within the current esa science programme lisa is in the cosmic vision 20152025 programme , and launch after 2020 seems likely . in 2007 the national research council report on the nasa beyond einstein program ( soon to become the physics of the cosmos program ) gave lisa the highest scientific ranking , and it has been rated very highly in the astro2010 decadal survey . however , as stated above , as of earlier this year ( 2011 ) esa is considering the feasibility of lisa as a single agency mission . several of the key technologies for lisa are being testing on the lisa pathfinder mission ( formerly smart-2 ) . lisa pathfinder will fly the lisa technology package ( ltp ) , which essentially consists of a downscaled version of one lisa arm compressed from 5 million km to 38 cm . the ltp arm contains two test masses ( an emitter and a receiver ) with a doppler link between them . the three main things it will measure are : the acceleration phase noise caused by the relative motion of the emitter and receiver from non - gravitational forces ; the readout noise ; and noise caused by the departure of the doppler link from the ideal scheme , due to the fact that we are not truly measuring the relative accelerations of two point particles , but instead a more complex system of multiple doppler links and extended masses . it is designed to test the accuracy of these to within an order of magnitude of that required by the full lisa . other aspects of the mission that will be tested are the discharging of the test masses , the caging and release of the masses following launch and the micro - newton thrusters . as much as possible lisa pathfinder is currently scheduled for launch in mid 2013 , after which it will orbit the l1 point , with a 180-day mission plan . lisa is the most advanced space - based project , but there exist concepts for at least two more detectors . decigo ( deci - hertz interferometer gravitational wave observatory ) [ 274 , 199 ] is a japanese project designed to fill the gap in frequency between ground - based detectors and lisa , i.e. the 0.110 hz band . it would have a similar configuration to lisa with three drag - free spacecraft , but have far shorter arm lengths at 1000 km . although still early in its design there are plans for two precursor technology demonstration missions ( decigo pathfinder and pre - decigo ) , with a the main mission having a launch date in the mid-2020s . a similar mission , in terms of the frequency band it seeks to cover , is the us big bang observer ( bbo ) ( see [ 122 , 124 , 167 ] for overviews of the proposal ) . one of its main aims will be to detect the stochastic background from the early universe , but it can also be used for high precision cosmology . the current configuration would consist of three lisa - like constellations of three spacecraft each , with 50 000 km arm lengths , and separated in their orbit by 120. the launch of this mission would be after decigo , but is designed to be 23 times as sensitive . at lower frequencies than lisa , 0.1 hz1 mhz , there are chinese proposals for superastrod ( super astrodynamical space test of relativity using optical devices ) . significant effort worldwide has been invested , and is continuing to be invested , in the development of both ground and spaced based gravitational - wave detectors . over the coming years , ground - based detectors will reach sensitivities that will enable the direct observation of gravitational waves as predicted by einstein s general theory of relativity and open the exciting new field of gravitational - wave astronomy . studying the gravitational - wave sky could radically change our understanding of the universe , expanding our knowledge of fundamental physics , cosmology and relativistic astrophysics . gravitational - wave observations will allow us to gaze into the heart of the most violent events in the universe , and will help answer some of the biggest questions , particularly in cosmology when combined with other astronomical observations .

significant progress has been made in recent years on the development of gravitational - wave detectors . sources such as coalescing compact binary systems , neutron stars in low - mass x - ray binaries , stellar collapses and pulsars are all possible candidates for detection . the most promising design of gravitational - wave detector uses test masses a long distance apart and freely suspended as pendulums on earth or in drag - free spacecraft . the main theme of this review is a discussion of the mechanical and optical principles used in the various long baseline systems in operation around the world ligo ( usa ) , virgo ( italy / france ) , tama300 and lcgt ( japan ) , and geo600 ( germany / u.k . ) and in lisa , a proposed space - borne interferometer . a review of recent science runs from the current generation of ground - based detectors will be discussed , in addition to highlighting the astrophysical results gained thus far . looking to the future , the major upgrades to ligo ( advanced ligo ) , virgo ( advanced virgo ) , lcgt and geo600 ( geo - hf ) will be completed over the coming years , which will create a network of detectors with the significantly improved sensitivity required to detect gravitational waves . beyond this , the concept and design of possible future third generation gravitational - wave detectors , such as the einstein telescope ( et ) , will be discussed .

Introduction Gravitational Waves Detection of Gravitational Waves Main Noise Sources Laser Interferometric Techniques for Gravitational-Wave Detectors Operation of First-Generation Long-Baseline Detectors Longer Baseline Detectors in Space Conclusion

PMC3662096

in fact , the indications for this surgical modality of common bile duct stone ( cbds ) have been limited with the advances and increasing experience in laparoscopic and endoscopic procedures . the advantages offered by this procedure , as well as its risks and disadvantages , also have been well noted in most of the already quoted bibliographic references . however , with three exceptions , the long - term results of choledochoduodenostomy ( cds ) , particularly in reference to the incidence of complications such as cholangitis , sump syndrome and risk of late development of cholangiocarcinoma have not been documented clearly . this retrospective study reviews the results of 51 consecutive cases of open biliary - enteric bypass carried out for cbds over a period of 5 years . our objective was to review the indications and outcome of this procedure in the endoscopic and laparoscopic era . we conducted a descriptive retrospective chart review of all the patients undergoing open bed for cbds in general surgery department at farhat hached hospital of sousse in tunisia between january 2005 and december 2009 . data were collected from patient 's medical folders with particular attention to perioperative course , symptoms , complications , hospital length of stay , and mortality . short - term outcomes were assessed through clinical reports at outpatient follow - up and hospital readmissions . the main surgical technique used was a side to side cds after washing systematically the cbd without verifying its clearance by a choledocoscope or a cholangiogram . the anastomosis was constructed with 4 - 0 vicryl suture with small atraumatic needles in a continuous running ( anterior and posterior rows ) or interrupted fashion : it was left to the surgeon 's discretion to select the type of suturing . ninety - three patients were treated for cbds with several procedures during the 5 years period . only 51 ( 40 women [ 78% ] and 11 men ) underwent open bed . the age range was 36 - 87 years with mean age 72 years ( standard deviation [ sd ] 9,7 ) . they presented with acute cholangitis ( 28% ) , abdominal pain ( 86% ) , jaundice ( 21% ) , acute pancreatitis ( 6% ) , and acute cholecystitis with asymptomatic cbd stone ( 6% ) . demographic and clinical details of 51 patients the main indications for biliary enteric bypass were elderly patients ( 90% were over 65 years old ) , multiple ( atleast 5 ) stones ( 54.9% ) and unextractable calculi ( 15.7% ) . the remaining patients had different clinical and intra - operative presentation : congenital biliary cyst ( 1 case ) , accidental intra - operative transection of cbd with transversal choldochotomy ( 1 case ) , cholcysto - choledochal fistulas ( 1 case ) , suspicious chronic pancreatitis associated with cbd calculi ( 1 young woman ) and a recurrent lithiasis in an old woman with permanent biliary stenting . we performed side to side cds in 49 patients ( 96% ) and end to side type in one patient who had iatrogenic transection of cbd . the cbd diameter was larger than 16 mm in 37.3% of the patients and 10 mm in 98% of the patients . post - operative complications [ table 2 ] occurred in 5 ( 10% ) patients , including one case of intra - abdominal abscess ( 2% ) successfully managed by transparietal drainage , wound infection ( 4% ) , two cases of heart failure ( 3.9% ) and one patient had post - operative peritonitis subordinate to ruptured liver abscess previously unnoticed , he was re - operated and had successful peritoneal toilet . post - operative deaths : two patients ( 3.9% ) died in the hospital during the same hospitalization for their cds . the first was an 80-year - old woman with cardiac history admitted for acute cholecystitis associated to asymptomatic cbds ; she died of post - operative heart failure . the second was a 76-year - old woman admitted for severe acute cholangitis with hemodynamic failure . post - operative complications the mean hospital stay was 10.9 days ( sd 6.5 ) and the mean post - operative stay was 7 days ( sd 3.8 ) . eight patients left the hospital in good condition and were lost to follow - up , so it is presumed that their intervention was initially successful . they never come to outpatient control . during the follow - up ( 1 - 6 years ) of the remaining patients , two had further intervention for right subcostal incisional hernia and one presented non - resectable head pancreatic cancer with multiple liver metastases 1 year after cds . our data corroborating others , as illustrated in table 3 , indicate that side to side cds is a very safe procedure with low morbidity and mortality . no long - term complications , as cholangitis , sump syndrome , or cholangiocarcinoma were observed . comparison of outcomes of different studies it should be noticed that we opted for bed because of not only its benefits mentioned further in the text but also the lack of interventional endoscopist and laparoscopic instruments particularly the helical basket in our hospital . indeed cds provides a safe and effective biliary drainage when carried out for carefully chosen indications . the procedure may still be required in selected cases , even with all the endoscopic and laparoscopic advances . we performed the side to side type of cds for 49 patients ( 96% ) including two young women ( 36- and 38-years - old ) as it is technically easier and faster than its end to side variety and hjs . it also permits to avoid the particular problem of residual stone . these criteria are capital when choosing among bed modalities particularly for high risk and elderly patients . furthermore , the latter with their limited life expectancy should not develop long - term complications . reports on laparoscopic cds have been published , but these procedures need considerable experience and expensive technologies . the two most notable objections to cds are the possibility of cholangitis and the presence of the symptoms produced by the so called sump / blind sac syndrome . thus many authors suggested a cbd diameter larger than 16 mm precaution to allow a cds with an opening no smaller than 14 mm in diameter.[17 ] our study , with no such complications , disproves this condition ( only 37% of patients had cbd diameter larger than 16 mm ) . moreover , de almeida ensured that a wide anastomosis can be constructed even on a duct 10 - 12 mm wide , and this concur with our results ( 98% of our patients had cbd 10 mm ) . such well performed cds prevents bile stasis , avoids the buildup of excessive intra - ductal pressure and permits a free flow of common duct contents including duodenal reflux as well as eventual retained / recurrent lithiasis back in the duodenum . the prevalence of blind sac syndrome is reported between 2.5% and 9.6% in several studies . it presumably derives from stasis and refluxed duodenal contents ( food , stones , or debris ) in to the terminal common duct , with bacterial overgrowth enhancing bile salt deconjugation . so factors leading to sump syndrome are a wide distal choledoque , papillary stenosis , retained stones , and anastomosis stricture . this phenomenon leads to diarrhea , as observed in the blind - loop syndrome arising anywhere else in the gut , can cause secondary liver abscess and facilitates deposition and reformation of calcium bilirubinate stones which could not be documented in spite of an exhaustive search . a very wide anastomosis could be the explanation.[810 ] in order to avoid this so feared syndrome , lasnier recommended end to side cds for young patient or in case of incomplete clearance of cbd and/or oddi dysfunction . otherwise they opted for side to side cds specially for elderly patients , in case of altered anatomy after billroth ii surgery , and when he faced up to technical difficulties due to inflammation , sclerosis and portal hypertension . however , mavrogiannis reported a considerably high recurrence rate 19% ( 6 patients/31 ) of the syndrome ( restenosis of the sphincterotomy opening ) after initially successful such management . it occurred 31 - 72 months ( median : 58.5 months ) after the initial treatment and new endoscopic treatment with a papillotomy was effective . in case of stoma stricture the best treatment is the endoscopic balloon cholangioplasty.[413 ] the blind sac syndrome associated to side to side hjs is extremely rare . indeed stricture is the main complication of hjs and rates , in most series , varies from 0% to 12% ( with most being around 8% ) and from 2.4 to 4.8% in case of biliary lithiasis . technique requirements for a good anastomosis are good submucosal apposition , single - layer closure , and mucosal inversion . in case of hepatolithiasis , bed results in high frequency of reflux cholangitis and thus should be abandoned . in recent years the number of reports of cholangiocarcinoma occurring in patients who previously had bed many years ago , has been increasing , so it has been suggested as a possible long - term complication of these surgical procedures . its incidence ranges from 2.8 - 7.4% at interval average of 10 years after choldechoenterostomy.[1720 ] the direct connection between the intestinal tract and biliary tree and reflux of activated pancreatic juice and intestinal bacteria into the biliary tract are considered as the factors causing chronic relapsing cholangitis , the latter has been suspected as a predisposing factor for the late development of cholangiocarcinoma . bed permits free communication between gastro - intestinal and biliary tracts , depriving the anti - reflux function of the sphincter of oddi . histologic examination of resected specimens , including cholangiocarcinoma of the biliary tract arising from long - standing cholangitis secondary to a bilioduodenal drainage , has shown , at sites distant from the malignancy , various degrees of hyperplastic lesions occurring in the area of cholangitis . this strengthens the hyperplasia - dysplasia - carcinoma progression hypothesis of a biliary carcinogenesis mechanism . the roux - en - y technique is the one expected to produce lower rates of cholangitis because it is immune to pancreatic reflux and less prone to intestinal bacterial backflow because of the protective action exerted by the interposed jejunal loop . tocchi reported a retrospective study of 1003 patients who underwent biliary enteric anastomosis ( transduodenal sphincteroplasty , cds , and hjs ) for benign disease with 129.6 years mean follow - up : univariate and multivariate analysis showed a significantly higher incidence of cholangiocarcinoma in the cds group ( p = 0.02 ) , and cholangitis was confirmed to be the only independent factor affecting its incidence ( p = 0.001 , odds ratio 35.7 ) . thus hjs should be considered straightaway for young patients in spite of cds benefits . they also noted that patients with such anastomosis had higher incidence of gastric lesions when compared to those with t - tube drainage or endoscopic sphincterotomy . the fact that one of our patients presented non - resectable and metastatic head pancreatic cancer 1 year after the cds , suggests that the tumor was present at the time of bd but was not detected . large retrospective studies and prospective randomized trials are needed to establish the definitive risk for late biliary carcinogenesis after bilio - intestinal drainage . thus we suggest that any patient treated with these procedures and experiencing relapsing cholangitis should be monitored for the late development of bile duct cancer . in case of asymptomatic cbs , we readily opt for minimally invasive techniques ( laparoscopy and/or endoscopy ) for young patients or fine cbd . indeed considering cds inconvenients , it was performed for only old patients with dilated cbd provided that surgery is nt considered inappropriate . open biliary drainage procedures may still indicated in select patients where the facility or expertise for minimally invasive biliary procedures is not available . side - to - side cds is a safe and highly effective therapeutic measure , even when performed on ducts less than 15 mm wide , provided a few technical requirements are respected . it should only be considered as obsolete after extensive , long - term , prospective , randomized assessments of laparoscopic or combined laparo - endoscopic approaches have been shown to be as effective as or superior to cds even in young population . patients experiencing relapsing cholangitis after bed should be closely monitored for the late development of biliary tract malignancies . the procedure should be regarded as an essential in the general surgical knowledge and training .

background : nowadays , biliary - enteric drainage ( bed ) is regarded as a last resort or obsolete therapeutic method for common bile duct stone ( cbds ) not only because of advances in minimally invasive therapeutic modalities but also due to fears of higher morbidity , cholangitis , and sump syndrome.aim:the present study aimed at evaluating the outcome of this procedure for choledocholithiasis.materials and methods : it is a retrospective review of 51 patients who underwent open choledochoenterostomy for cbds between january 2005 and december 2009.results:about 40 women ( 78% ) and 11 men underwent open bed ( mean age 72 years ) . indications were elderly patients ( 90% ) , multiple stones ( 54.9% ) and unextractable calculi ( 15.4% ) . we performed 49 ( 96% ) side to side choledochoduodenostomies , one end to side choledochoduodenostomy ( cds ) and one end to side hepaticojejunostomy . the mortality rate was 3.9% . overall morbidity was 12% with no biliary leakage . with a decline of 1 - 6 years , neither sump syndrome nor cholangiocarcinoma occurred.conclusions:side-to-side cds is a safe and highly effective therapeutic measure , even when performed on ducts less than 15 mm wide , provided a few technical requirements are respected . patients experiencing relapsing cholangitis after bed should be closely monitored for the late development of biliary tract malignancies .

Introduction Materials and Methods Results Discussion Conclusions

PMC4621650

implementation of a fair , systematic , and reliable evaluation system of the faculty members activities is a challenging issue . this evaluation is mostly performed in the areas of teaching , research and innovation , professional practice , commitment , and citizenship and should be able to show the interpersonal differences in the above - mentioned areas . it would help universities to make decisions regarding promotion , appointment , faculty compensation , recruiting , granting tenure , and rewarding excellence based on objective criteria . on the other hand , expectations and the evaluation criteria should be clear and announced to the faculty members . in spite of the global interest for these kinds of evaluation systems , few studies have assessed systems that can cover all the activities of the faculty members . this system should be able to differentiate among faculty members , observe the mission of the university , and be applicable to all faculty members . some medical schools started to evaluate the activities of their faculty members based on mission based management ( mbm ) presented by association of american medical colleges and computer sciences corporation in the late 1990s . mbm emerged as a way to understand the costs and revenues associated with the multiple missions of the medical school ; align faculty members activities with the school 's mission ; provide transparent data ; and make decisions based on those data . evaluation of the activities and productivity of the faculty members , especially in education , is very complicated ; however , several solutions have been proposed including the experiences of the universities of dalhousie , texas health science center , and wisconsin . moreover , several papers have been published in this regard since 1995 . medical school of tehran university of medical sciences ( tums ) implemented an objective metrics system to evaluate the faculty members performance quantitatively in the areas of education , research , and service provision in 2009 . this system is called shoa , a persian abbreviation for a phrase meaning academic performance metrics and valuing and is designed based on mbm concepts . in this system , the activities of faculty members are arranged in five categories including education ( theoretical teaching , teaching in laboratories and practice , clinical and field training , educational workshops , consultation and supervision , evaluation , educational products , research in education , and self - promotion ) , research ( research projects , articles and research products , research workshops and consultation and supervision ) , clinical services ( patient care and service providing with and without presence of students ) , administrative affairs ( management positions and participation in official meetings ) , and out of university academic activities . data are collected through a web - based software in which each faculty member has a password protected home page . faculty members self - report their activities along with their details during each evaluation period ( 1-month ) and at the end send them to a verifier who is either department 's or clinical ward 's dean . the verifier can return some data to the faculty member for more explanation or correction . each activity has a relative value scale based on the time required for preparation and presentation , group or individual nature of the activity and its importance . a relative value unit ( rvu ) after 5 years , the coverage of the shoa system is 79% and 75% in clinical and basic sciences departments respectively . some applications of the system include determining the expectancies from faculty members , directing the activities of the faculty members and educational departments , promotion , and assessment of the effective physical presence . there is no evidence - based structured model for faculty members metrics system in other universities as well . therefore , we decided to meta - evaluate the shoa system to devise a model for evaluation of the activities of the faculty members . for meta - evaluation , the personal evaluation standards presented by joint committee on standards for educational evaluation were used . these standards are provided in four areas of propriety standards ( 7 standards ) , utility standards ( 6 standards ) , feasibility standards ( 3 standards ) , and accuracy standards ( 11 standards ) . this mix method study with explanatory design and qual - quan approach was performed in tums medical school in 2014 . the school has 10 basic sciences and 25 clinical departments with 99 ( 10% ) and 884 ( 90% ) faculty members , respectively . then we conducted semi - structured interviews with the faculty members regarding joint committee meta - evaluation standards . then , through content analysis of the interviews , we designed a questionnaire which was completed online by faculty members . in the final stage , using questionnaire 's factor analysis , the components of the model of the evaluation of the faculty members activities were determined . the validity of the system had been evaluated previously during developing shoa by reviewing the activities list by all department deans ( 35 deans ) to ensure complete coverage of all faculty members activities . to evaluate the reliability of the system , we test - retested self - reported data for september 2013 . forty faculty members were randomly selected proportionate to size from basic sciences and clinical departments . four of them who had not entered their data in september 2013 and two who had refused to participate were excluded . 1-month after the evaluation period , we saved and removed the entered information off the system and asked the participants to re - enter their activities . the reliability of the system was calculated through assessing the correlation of the rvus of the activities in the two stages . to evaluate the stance of regarding joint committee meta - evaluation standards in the shoa system , we conducted semi - structured interviews with faculty members who had at least 2 years of experience with the shoa . a same interviewer conducted all interviews and asked the faculty members to discuss issues such as how to provide a list of academic activities and their values , how to increase the validity and reliability of the system , the process of evaluation and data verification , and how obtained information could be utilized . two experts performed content analysis and the results were compared to achieve trustworthiness . in the next stage , according to the codes and themes extracted from the interviews , we designed a questionnaire . the questionnaire included 13 demographic and 33 likert - type main questions ranging from 0 ( very little ) to 4 ( very much ) . evaluation of the reliability with retest after 2 weeks in a sample of 25 faculty members showed a correlation coefficient ( r ) of 0.81 . finally , three sources were used to design the model : the results of the factor analysis of the questionnaire , the results of interviews content analysis , anda focus group with the participation of medical education experts for finalizing the model . the results of the factor analysis of the questionnaire , the results of interviews content analysis , and a focus group with the participation of medical education experts for finalizing the model . as the study is a mix method one , we first present the results of quantitative and qualitative parts separately and then introduce the draft and final model . comparison of the rvus obtained from test and retest using paired t - test showed no significant difference . reliability of faculty members activities metrics system in medical school we asked interview participants questions relating to eight domains of the academic activities list , activities values , validity , reliability , workflow , data verification , application of information , and effects of system in faculties performance . using qualitative content analysis , we coded the transcribed text and extracted 96 subthemes from which 65 items remained after omitting phrases with overlapping statements . then we reviewed these 65 subthemes to put relevant concepts in a same theme with an appropriate name . finally , we reached 10 main themes which were used as the factors of the final model . main themes with some of their relevant subthemes resulted from qualitative content analysis of interviews also , interviews showed that the status of meta - evaluation standards was acceptable except for the standard of functional reporting in the area of utility standards which was not met . functional reporting meant that reports should be clear , timely , accurate , and germane so that they were of practical value to the evaluates and other appropriate audiences . a total number of 488 faculty members completed the questionnaire ( response rate = 49.6% ) . since , the faculty members were informed for participation in the survey by their academic email addresses and many of them used other email services , it seems that the corrected response rate could be higher . table 3 shows the characteristics of the participants in the survey whose mean age was 45 ( 7.9 ) years . demographic characteristics of the faculty members activities metrics system survey participants in medical school we used exploratory factor analysis to extract main factors . kaiser - mayer - olkin index was 0.914 , and bartlett 's test of sphericity was 5238.162 ( p < 0.001 ) that showed the high adequacy of the items and its suitability for factor analysis . to extract factors , we used varimax rotation method with four , five , and six factors solutions . finally , it seemed that the six factors solution was the most suitable one . in this solution , eigenvalues were greater than one and could explain 66.20% of the variability . eight questions which were not loaded in these six factors and their removal had no significant effect on the variance explanation were removed . table 5 shows the factors labels with their cronbach 's alpha , mean and standard deviation . since , the average of all factors was above 2 ( the midpoint of the likert scale ) , they were all used in designing the model . labels and related questions of extracted factors of faculty members activities metrics system survey characteristics of extracted factors of faculty members activities metrics system survey the model draft had six dimensions and nine factors . the dimensions of the model should be considered in the whole evaluation system of the faculty members activities and were mainly derived from factor analysis of the questionnaire . factors included the executive components of the system and were mainly obtained via qualitative content analysis of the interviews with faculty members . the dimensions of the model draft included : mission alignment the evaluation system of the faculty members activities should be effective in promoting their performance . the performance should cover the school 's missions including education , research , clinical services , and administrative and managerial affairs . . the system should be able to help the faculty members and managers to identify performance weaknesses and strengths . moreover , it should be able to identify the excellent performance.accuracy the information derived from the system should be accurate as much as possible . the list of the activities should cover all academic affairs , and their value should be close to their real ones.explicit faculty members should receive adequate information on how to complete the required data , evaluation results usages , and guidelines , and bylaws . they should also be informed on the clear definition of the activities listed in the system.satisfaction faculty members should be satisfied with the way their questions and ambiguities regarding the system are answered , and the way and time of verifying their self - reported data.appropriateness the minimum expectancies from faculty members in the areas of education , research , and clinical services should be appropriate.constructiveness the evaluation system should encourage faculty members to perform activities on the list that they did not do and , on the other hand , keep from activities that they previously did but are not included in the list because they are not in line with the missions . the system should also encourage faculty members to modify their activities according to the system . mission alignment the evaluation system of the faculty members activities should be effective in promoting their performance . the performance should cover the school 's missions including education , research , clinical services , and administrative and managerial affairs . the system should be able to help the faculty members and managers to identify performance weaknesses and strengths . accuracy the information derived from the system should be accurate as much as possible . the list of the activities should cover all academic affairs , and their value should be close to their real ones . explicit faculty members should receive adequate information on how to complete the required data , evaluation results usages , and guidelines , and bylaws . they should also be informed on the clear definition of the activities listed in the system . satisfaction faculty members should be satisfied with the way their questions and ambiguities regarding the system are answered , and the way and time of verifying their self - reported data . appropriateness the minimum expectancies from faculty members in the areas of education , research , and clinical services should be appropriate . constructiveness the evaluation system should encourage faculty members to perform activities on the list that they did not do and , on the other hand , keep from activities that they previously did but are not included in the list because they are not in line with the missions . the system should also encourage faculty members to modify their activities according to the system . the executive factors of the model draft included : consensus the list of the activities and their values should be prepared upon the consensus of all the faculty members . the activities should be revised independently in each department and customized accordingly.self-reporting the activities should be self - reported and then verified by an informed and acceptable person.web-based system data entry of the activities , their verification and correction , data analysis , and notifications should be done through a web - based software.evaluation period faculty members activities data should be entered in the system in the predefined evaluation period , preferably 1-month.minimum expectancies the expected rvus from faculty members in the areas of education and research should be clear and announced formally.analysis intervals although the evaluation period was 1-month , the mean rvus for each faculty members should be calculated in 1-year intervals . in this way , different amounts of activities in different months did not cause any problems in the evaluation.verifiers each verifier should be in charge of verifying the data of a maximum of 15 faculty members to increase the accuracy and speed of the process.flexibility all the components of the evaluation system including the list of activities , their values , minimum expectancies , and workflow should be flexible and revised every 1 or 2 years.decision making the scores ( rvus ) obtained from the system should not replace the school administrators decisions and are only used to help them in the process of decision making.validity the validity of the system should be constantly evaluated . for this reason , the documents and evidences of the activities of a number of randomly selected faculty members would objectively be assessed . consensus the list of the activities and their values should be prepared upon the consensus of all the faculty members . self - reporting the activities should be self - reported and then verified by an informed and acceptable person . web - based system data entry of the activities , their verification and correction , data analysis , and notifications should be done through a web - based software . evaluation period faculty members activities data should be entered in the system in the predefined evaluation period , preferably 1-month . minimum expectancies the expected rvus from faculty members in the areas of education and research should be clear and announced formally . analysis intervals although the evaluation period was 1-month , the mean rvus for each faculty members should be calculated in 1-year intervals . in this way , different amounts of activities in different months did not cause any problems in the evaluation . verifiers each verifier should be in charge of verifying the data of a maximum of 15 faculty members to increase the accuracy and speed of the process . flexibility all the components of the evaluation system including the list of activities , their values , minimum expectancies , and workflow should be flexible and revised every 1 or 2 years . decision making the scores ( rvus ) obtained from the system should not replace the school administrators decisions and are only used to help them in the process of decision making . for this reason , the documents and evidences of the activities of a number of randomly selected faculty members would objectively be assessed . a focus group was held with the participation of the medical school dean , three medical education experts , and four faculty members who were experienced in the area of evaluation to finalize the above - mentioned model . in this session which took 4 h , all the factors of the model were discussed and reviewed . since , the components of the model were directly extracted from the views of the faculty members , caution was exercised not to change or modify the items only based on taste . the only modification was removing validity from factors since the members of the focus group believed that it was part of accuracy and did not require a separate entity . comparison of the rvus obtained from test and retest using paired t - test showed no significant difference . we asked interview participants questions relating to eight domains of the academic activities list , activities values , validity , reliability , workflow , data verification , application of information , and effects of system in faculties performance . using qualitative content analysis , we coded the transcribed text and extracted 96 subthemes from which 65 items remained after omitting phrases with overlapping statements . then we reviewed these 65 subthemes to put relevant concepts in a same theme with an appropriate name . finally , we reached 10 main themes which were used as the factors of the final model . main themes with some of their relevant subthemes resulted from qualitative content analysis of interviews also , interviews showed that the status of meta - evaluation standards was acceptable except for the standard of functional reporting in the area of utility standards which was not met . functional reporting meant that reports should be clear , timely , accurate , and germane so that they were of practical value to the evaluates and other appropriate audiences . a total number of 488 faculty members completed the questionnaire ( response rate = 49.6% ) . since , the faculty members were informed for participation in the survey by their academic email addresses and many of them used other email services , it seems that the corrected response rate could be higher . table 3 shows the characteristics of the participants in the survey whose mean age was 45 ( 7.9 ) years . demographic characteristics of the faculty members activities metrics system survey participants in medical school we used exploratory factor analysis to extract main factors . kaiser - mayer - olkin index was 0.914 , and bartlett 's test of sphericity was 5238.162 ( p < 0.001 ) that showed the high adequacy of the items and its suitability for factor analysis . to extract factors , we used varimax rotation method with four , five , and six factors solutions . finally , it seemed that the six factors solution was the most suitable one . in this solution , eigenvalues were greater than one and could explain 66.20% of the variability . eight questions which were not loaded in these six factors and their removal had no significant effect on the variance explanation were removed . table 5 shows the factors labels with their cronbach 's alpha , mean and standard deviation . since , the average of all factors was above 2 ( the midpoint of the likert scale ) , they were all used in designing the model . labels and related questions of extracted factors of faculty members activities metrics system survey characteristics of extracted factors of faculty members activities metrics system survey the dimensions of the model should be considered in the whole evaluation system of the faculty members activities and were mainly derived from factor analysis of the questionnaire . factors included the executive components of the system and were mainly obtained via qualitative content analysis of the interviews with faculty members . the dimensions of the model draft included : mission alignment the evaluation system of the faculty members activities should be effective in promoting their performance . the performance should cover the school 's missions including education , research , clinical services , and administrative and managerial affairs . the system should be able to help the faculty members and managers to identify performance weaknesses and strengths . moreover , it should be able to identify the excellent performance.accuracy the information derived from the system should be accurate as much as possible . the list of the activities should cover all academic affairs , and their value should be close to their real ones.explicit faculty members should receive adequate information on how to complete the required data , evaluation results usages , and guidelines , and bylaws . they should also be informed on the clear definition of the activities listed in the system.satisfaction faculty members should be satisfied with the way their questions and ambiguities regarding the system are answered , and the way and time of verifying their self - reported data.appropriateness the minimum expectancies from faculty members in the areas of education , research , and clinical services should be appropriate.constructiveness the evaluation system should encourage faculty members to perform activities on the list that they did not do and , on the other hand , keep from activities that they previously did but are not included in the list because they are not in line with the missions . the system should also encourage faculty members to modify their activities according to the system . mission alignment the evaluation system of the faculty members activities should be effective in promoting their performance . the performance should cover the school 's missions including education , research , clinical services , and administrative and managerial affairs . the system should be able to help the faculty members and managers to identify performance weaknesses and strengths . accuracy the information derived from the system should be accurate as much as possible . the list of the activities should cover all academic affairs , and their value should be close to their real ones . explicit faculty members should receive adequate information on how to complete the required data , evaluation results usages , and guidelines , and bylaws . they should also be informed on the clear definition of the activities listed in the system . satisfaction faculty members should be satisfied with the way their questions and ambiguities regarding the system are answered , and the way and time of verifying their self - reported data . appropriateness the minimum expectancies from faculty members in the areas of education , research , and clinical services should be appropriate . constructiveness the evaluation system should encourage faculty members to perform activities on the list that they did not do and , on the other hand , keep from activities that they previously did but are not included in the list because they are not in line with the missions . the system should also encourage faculty members to modify their activities according to the system . the executive factors of the model draft included : consensus the list of the activities and their values should be prepared upon the consensus of all the faculty members . the activities should be revised independently in each department and customized accordingly.self-reporting the activities should be self - reported and then verified by an informed and acceptable person.web-based system data entry of the activities , their verification and correction , data analysis , and notifications should be done through a web - based software.evaluation period faculty members activities data should be entered in the system in the predefined evaluation period , preferably 1-month.minimum expectancies the expected rvus from faculty members in the areas of education and research should be clear and announced formally.analysis intervals although the evaluation period was 1-month , the mean rvus for each faculty members should be calculated in 1-year intervals . in this way , different amounts of activities in different months did not cause any problems in the evaluation.verifiers each verifier should be in charge of verifying the data of a maximum of 15 faculty members to increase the accuracy and speed of the process.flexibility all the components of the evaluation system including the list of activities , their values , minimum expectancies , and workflow should be flexible and revised every 1 or 2 years.decision making the scores ( rvus ) obtained from the system should not replace the school administrators decisions and are only used to help them in the process of decision making.validity the validity of the system should be constantly evaluated . for this reason , the documents and evidences of the activities of a number of randomly selected faculty members would objectively be assessed . consensus the list of the activities and their values should be prepared upon the consensus of all the faculty members . self - reporting the activities should be self - reported and then verified by an informed and acceptable person . web - based system data entry of the activities , their verification and correction , data analysis , and notifications should be done through a web - based software . evaluation period faculty members activities data should be entered in the system in the predefined evaluation period , preferably 1-month . minimum expectancies the expected rvus from faculty members in the areas of education and research should be clear and announced formally . analysis intervals although the evaluation period was 1-month , the mean rvus for each faculty members should be calculated in 1-year intervals . in this way , different amounts of activities in different months did not cause any problems in the evaluation . verifiers each verifier should be in charge of verifying the data of a maximum of 15 faculty members to increase the accuracy and speed of the process . flexibility all the components of the evaluation system including the list of activities , their values , minimum expectancies , and workflow should be flexible and revised every 1 or 2 years . decision making the scores ( rvus ) obtained from the system should not replace the school administrators decisions and are only used to help them in the process of decision making . validity the validity of the system should be constantly evaluated . for this reason , the documents and evidences of the activities of a number of randomly selected faculty members would objectively be assessed . a focus group was held with the participation of the medical school dean , three medical education experts , and four faculty members who were experienced in the area of evaluation to finalize the above - mentioned model . in this session which took 4 h , all the factors of the model were discussed and reviewed . since , the components of the model were directly extracted from the views of the faculty members , caution was exercised not to change or modify the items only based on taste . since the members of the focus group believed that it was part of accuracy and did not require a separate entity . conceptual models are employed to better identify and understand the phenomena that are conceptualized in the brain and can be used for structuration of a managerial problem in order to summarize the views of the experts on what is right . in this study , we evaluated a system that was operational for some years to present a model from the viewpoint of its main users , that is , faculty members . therefore , we determined its validity and reliability . then we investigated the views of the faculty members through qualitative interviews , and a questionnaire was designed based on the results of the interviews . joint committee personnel evaluation standards were observed in all the stages . in the end , the model was presented considering the results of the content analysis of the interviews , factor analysis of the questionnaire , and experts opinion through a focus group . one of the important points affecting this study is how the shoa system has been installed and operated in tums . the little resistance to its installation , when the system was designed , has faded over time since the system is flexible and revised periodically . the shoa represents the views of the faculty members who have used it in the past years . for this reason , it was not surprising that the views of the faculty members were close to the running system , and in qualitative interviews , data were saturated after about the 10 interview , though we continued to 18 interviews to cover different educational groups . joint committee personnel evaluation standards were acceptable , especially the validity and reliability of the system which were higher than expectations . only the standard functional reporting in the area of utility standards was not met . therefore , after the project , a module was added to the system that enabled the faculty members to evaluate their activities in a period of time of their choice and calculate their own rvus based on different categories . the majority of the reports in universities with similar systems concerns and explains the installation of the system , determining the list of activities , and their weights ; and mainly focuses on multiple source decision making and analysis of the obtained data . in this study , we tried to present a model based on the viewpoints of the faculty members considering the 5-year experience of the shoa system . the main objective of mbm is to align the activities of the faculty members to the school missions . from the standpoint of faculty members , the evaluation system should differentiate between clinical and basic sciences faculty members fairly . unlike basic sciences faculty members , clinical faculty members obtain a great proportion of their rvus through clinical activities . therefore , evaluation of activities should be based on the spectrum of mission - aligned activities in each department ; otherwise , the results always falsely show that some departments are less active than others . this concern mostly exists for educational activities and is resolved by the factor of minimum expectancies in the model ; in other words , calculation of minimum rvus expected from faculty members for educational activities is performed independently in each department . in the shoa , educational expectancy calculation is norm - referenced for each department , and the mean minus one standard deviation is announced as the mean expected rvus for that department . on the other hand , research activities , the faculty members insisted that managerial activities should not be included in the minimum expectancies and the rvus obtained from administrative affairs should only be included in the total score . as a result , for the promotion of faculty members , three domains of education , research , and total scores should be independently evaluated . due to the insistence of the faculty members in this regard , appropriateness the dimensions of accuracy , explicit , and satisfaction are very close to personnel evaluation standards and were specially emphasized by faculty members . one of the considerations in the evaluation of faculty members is its effect on their performance or being constructive . the faculty members also focused on the importance of the issue which was discussed under constructiveness . the faculty members in this study put emphasis on cooperation in different stages of designing the evaluation system , especially preparing the list of activities and their values , which was added as the factor of consensus to the model . moreover , constant correction and modification of the system proportionate to changes in programs and job descriptions of departments were another demand of the faculty members which were addressed as flexibility in the model factors . self - reporting and the role of verifiers were other discussed issues . the faculty members believed that they should be trusted although they insisted on verification of their self - reported activities with consideration of their respected position . having a web - based evaluation system to make the process easier was one of the discussed issues . although data entry intervals were 1-month , calculation of obtained scores for decision making was based on the monthly average of their activities through a year . since activities vary in different months , their monthly average provides a better estimate of the activities of the faculty members in 1-year . finally , the faculty members expected the school managers to evaluate their activities intelligently and to use the shoa results as an assistant in the process of decision making rather than making decision only based on obtained rvus . in this study , the questionnaire was mailed only to participants academic email address that could be considered as a limitation since some of them may use other email services . the model in this study was only presented based on the viewpoints of the faculty members as evaluates ; therefore , other stakeholders may have other dimensions and factors in mind which should be investigated in future studies . moreover , we did not investigate how the list and weight of the activities were determined , what were the problems of collecting the data of the faculty members activities especially educational ones , and how the quality of the activities and its relationship with their quantity should be assessed , which require further studies . in this study , we evaluated a faculty members performance metrics system and investigated faculty members views through qualitative interviews and a quantitative survey . self - reported faculty members activities had acceptable reliability and validity and would be used for decision making . the proposed model for faculty members activities evaluation consisted of six dimensions : mission alignment , accuracy , explicit , satisfaction , appropriateness , and constructiveness and nine executive factors : consensus , self - reporting , web - based system , evaluation period , minimum expectancies , analysis intervals , verifiers , flexibility , and decision making and could be used for designing and implementing such systems in medical schools . am contributed in the conception and design of the work and acquisition , analysis , and interpretation of data , conducting the study , drafting and revising the manuscript , approval of the final version of the manuscript , and agreed for all aspects of the work . ksa contributed in the design of the work , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . rm contributed in the design of the work , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . mj contributed in the design of the work , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . hkv contributed in the design of the work , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work .

background : there is a global interest for deploying faculty members activities evaluation systems , however implementing a fair and reliable system is a challenging issue . in this study , the authors devised a model for evaluation of faculty members activities with regard to their viewpoints and meta - evaluation standards.materials and methods : the reliability of the current faculty members activities metrics system was investigated in medical school of tehran university of medical sciences in 2014 . then authors conducted semi - structured interviews regarding meta - evaluation standards and designed a questionnaire based on interviews results which were delivered to faculty members . finally , they extracted the components of the model regarding interviews content analysis and questionnaire 's factor analysis and finalized them in a focus group session with experts.results:reliability of the current system was 0.99 ( p < 0.05 ) . the final model had six dimensions ( mission alignment , accuracy , explicit , satisfaction , appropriateness , and constructiveness ) derived from factor analysis of the questionnaire and nine factors ( consensus , self - reporting , web - based system , evaluation period , minimum expectancies , analysis intervals , verifiers , flexibility , and decision making ) obtained via qualitative content analysis of the interviews.conclusion:in this study , the authors presented a model for faculty members activities evaluation based on meta - evaluation of the existing system . the model covered conceptual and executive aspects . faculty members viewpoints were the core component of this model , so it would be acceptable in a medical school to use the model for evaluating their activities .

INTRODUCTION MATERIALS AND METHODS RESULTS Reliability Interviews Questionnaire Designing the draft of the model Finalizing the model DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest AUTHOR'S CONTRIBUTION

PMC4696353

gap formation at cavity wall - restorative material interface is always of great importance , because of its significance to microleakage , postoperative sensitivity and secondary caries formation . bond strength and polymerization shrinkage of restorative materials are directly related to their adaptation to cavity walls through their flow , modulus of elasticity and wettability . cavity factors such as number of walls , access to the walls and wall quality , along with operator factors such as material type , material handling and method of insertion are also very important to wall adaptation . if these factors are not properly handled , a gap of variable size will be formed between cavity walls and filling material , during or following the hardening of the material . light , scanning electron , laser and confocal microscopy , dyes , tracers , air - pressure , electrical conductance and optical coherence tomography have all been used in the investigation of this gap . most of the above methods measure representative two - dimensional sections of cavities , interpolating the results for three - dimensional estimations . although , efforts in measuring the three - dimensional space in small cavities ( 2 mm in diameter ) were made , in minimally invasive interventions for caries , the question of how well a material can be adapted to the walls of cavities with smaller diameters ( 1.0 - 1.5 mm ) , still remains . opdam et al . found that voids and wall gaps were greater for thicker in consistency materials than medium or thin materials . in another study , olmez et al . found that internal voids correlated with marginal microleakage in class ii composite restorations . since material voids are equally important to gap space , and both are depended on the manipulation of the material , it is important to see if voids are also affected by the size of the cavity , during clinicians effort to insert the material to the bottom of the cavity . in the study of lioumi , it was noted that the width of material - cavity interface was not uniformly distributed over the entire cavity walls but it was different in different parts of the cavity , due to certain parameters . the widest gap formed at the internal line angles of the cavity when margins were beveled , etched and bonded , while a significant amount of voids appeared within the material close to or in contact with the interface . investigating the effect of configuration factor on gap formation in composites found that high c - factor values generate large gap formation . x - ray computed micro - tomography ( micro xct ) is a method that uses x - rays to create two - dimensional density images of the specimen 's cross sections and then reconstruct the specimen in a three - dimensional model . for this reason , it is very useful in many areas in dentistry including the investigation of material adaptation to cavity wall and margins , with relative accuracy , visualizing the different in density entities of the specimens . until now , a number of studies have been published on the adaptation of several different composite materials on cavity walls . however , most of these studies were focused on polymerization shrinkage in cavities of normal dimensions and the attention was given to a rather laboratory technique in order to facilitate measurements . for these reasons , a method that could permit the investigation of gaps in a more clinically relevant setting of the entire cavity preparation , and in dimensions closer to a very conservative cavity would be of significant importance to the study of gap formation and location in such restorations . the purpose of this study was to validate a microtomographic procedure for the comparison of voids ( densities ) in different ultraconservative cavities and to test the hypotheses that : the materials used for direct restoration of the cavities present no differences in the amount of internal voids or at the material - wall interface , voids and gaps are not related to cavity dimension , andlocation of voids is not dependent to a specific restorative material . the materials used for direct restoration of the cavities present no differences in the amount of internal voids or at the material - wall interface , voids and gaps are not related to cavity dimension , and location of voids is not dependent to a specific restorative material . for the purpose of this study , 12 human molar teeth were used for the preparation of 48 cylindrical cavities and their restoration with four different materials . the required sample size at a = 0.05 , a power of 0.85 and an effect size of 0.6 , was a priori computed using g power v.3.1.5 ( franz ful , universitat kiel , germany ) . visually intact teeth were carefully selected from recently extracted teeth , without the presence of hypomineralized or hypolplastic areas , caries or fracture lines on their buccal or lingual surfaces . teeth were kept continually in tap water until cavity preparations . in order to prepare cavities , as much as possible on the same horizontal tooth plane , the roots of all teeth , 2 - 3 mm below their cemento - enamel junction , were cut and removed . two buccal ans two linguall cavities of 2.5 mm in depth were prepared in each tooth under air - water spray , with a diamond bur rotating in a high - speed hand piece . two of the cavities were opened with a 0.8 mm in diameter bur ( fgd0 , strauss and co ) and two with a 1.0 mm diameter ( fgd1 , strauss and co ) . cavity depth was established with a length mark on each bur and cutting direction was facilitated by guiding the tooth against a stabilized hand piece with a horizontally rotating bur [ figure 1 ] . no special attention was given for opening the cavities with an exact width or depth , in order to have a range of cavities with differences in width or depth and facilitate their correlation with restorations gaps and voids . only four cavities were allowed to be opened by the same bur and cavities of different diameter were distributed equally to labial and lingual surfaces . after preparation of the cavities , all teeth were stored in water of 37c until their first microtomographic examination by skyscan 1072 microtomographer ( micro xct , model 1072 , skyscan aartselaar , belgium ) . the cavities were examined before insertion of the filling material , to calculate the precise cavity dimensions and assist the estimation of gaps and voids in the restored cavities at the second microtomographic examination [ figure 2 ] . operating parameters used for skyscan 1072 microtomographer upper left : microtomographic section of a tooth showing the prepared cavities . lower : a screen image showing the binarization settings with tview software , before calculation starts . the restorative materials used to fill the experimental cavities are shown in table 2 , and the procedure followed for each material is described below . the assignment of restorative material and bur diameter on tooth surfaces was based on a latin square design , in order to have teeth , with all the designed treatment variables , in a systematic rather than a random fashion . all cavities were acid etched with 37% ortho - phosphoric acid for 30 s , rinsed with water for 2 - 3 s and dried with air for 4 - 5 s and 1 - 2 absorbent paper points . cavities assigned to receive composite resins were lined carefully with adhesive according to manufacturer instructions ( prime and bond nt , dentsply int inc , milford , usa ) and polymerized for 20 s , using a curing unit with a light intensity of 550 - 600 mw / cm ( demetron lc / kerr gmbh , rastatt , germany ) . it must be mentioned that adhesive material was used with caution since excess of adhesive remain undetected by x - rays and even a small excess could be measured as gap . materials used in the study aeliteflo / bisco inc , chicago , usa ( af ) was placed in the cavity with a small diameter tip on material syringes . the tip was initially placed at the bottom of the cavity and was gradually withdrawn in an outward direction as the material was injected into the cavity . the material was polymerized for 30 s , and the excess , was finished and polished using medium , fine and superfine sof - lex discs ( 3 m espe , dental products , st . permaflo / ultradent products inc , stlouis / usa(pf ) was placed in the cavity exactly as af . aelite aesthetic / bisco inc , chicago , usa ( aa ) was placed in the cavity in small parts and condensed with the use of a dentin excavator , modified to a small diameter condenser ( 0.8 mm ) , in order to enter the cavities . after condensation and polymerization , the same finishing and polishing procedure as in af was followed . fuji ix / gc corporation , tokyo , japan ( fu ) was inserted in the cavity after activation of the capsule according to manufacturer 's instructions , by placing the capsule tip at the entrance of the cavity and condensing the material with the use of the same condenser , as before . immediately after finishing and polishing of restorations , teeth were placed in distilled water of 37c and retrieved after 24 h to receive 500 thermal cycles ( 5 - 55c , 3 min each ) . the second x - ray microtomographic examination was made under the same operating parameters with the first examination , immediately after thermocycling procedure . the volume amount of empty spaces within the material or between material and cavity walls was calculated on this second series of digital sections , using the tview v.1.3 software ( skyscan n.v . the software calculates on binary images of sample 's tomographic sections , the different in density areas , within a predefined region of interest ( roi ) . running all relative sections , the total volume ( vt ) and the volume fraction percent ( vf % ) of the interested densities ( called active pixels ) in the roi can be calculated . a specific process had to be followed , to ensure that all calculations were based exactly on the same roi . aartelaar , belgium , the number of horizontal sections covering the width of the cavity was determined . the sections showing all four cavities at their largest width were located and copied on a photoshop cs file . a circular area over the cavity was drawn on a transparent layer with the marquee tool . then , the circular marquee line was copied in new transparent layers , moved and applied to all four cavities for an exact positioning and all four layers were combined and saved as a single .bmp file . this process was repeated for all tooth cavities [ figure 2 ] and the saved .bmp files were placed as the last tomographic image , in order to remain available , but outside the range of the selected sections . the inserted image in each set of tooth microtomographs was located and the roi was drawn over the selected areas . the upper and lower sections of each cavity were inserted , and the color palette was set to binary colors ( 247 in level-1 and 255 in level-2 ) , for best discrimination of empty spaces from tooth / material substance [ figure 2 ] . the three - dimensional analysis was used , in order to collect information from the set of predefined horizontal sections on the variables described below . this three - dimensional analysis was repeated three times for each cavity and for all teeth . data collected concerned the following morphometric values : vt , the total selected volume throughout all the cross sections , vf % , the volume percent of the selected item ( empty space ) in an area , st , the total surface area of the item . the interest was centered on vf % and its relation to the initial volume of cavity dimensions and restorative material type . vt , the total selected volume throughout all the cross sections , vf % , the volume percent of the selected item ( empty space ) in an area , st , the total surface area of the item . the interest was centered on vf % and its relation to the initial volume of cavity dimensions and restorative material type . the location of voids and gaps was evaluated using the tview software and its no1 color palette . all relative to each cavity .bmp images ( 1024 1024 pixels ) were viewed by two examiners for the location of voids if present , on a 19 computer screen . for their classification three locations were recorded : the material itself , the side walls of the cavity andthe bottom of the cavity . voids at the walls or bottom of the cavity were actually the gaps at the material / cavity interface . for each cavity , one , two or all three locations were recorded , always based on the agreement of both examiners . voids at the walls or bottom of the cavity were actually the gaps at the material / cavity interface . for each cavity , one , two or all three locations were recorded , always based on the agreement of both examiners . kolmogorov - smirnov tests for normality and bartlett 's test for the equality of variances indicated the use of kruskal - wallis nonparametric test to analyze the data for differences between materials , in respect to the morphometric parameters . mann - whitney test was also used to search for possible differences between low and high viscosity materials , for each morphometric parameter . the degree of correlation of cavity dimensions with all morphometric parameters was analyzed by spearman 's test . all statistical tests performed with medcalc v.10.0 ( medcalc software , mariakerke , belgium ) at a 0.05 level of significance . for the purpose of this study , 12 human molar teeth were used for the preparation of 48 cylindrical cavities and their restoration with four different materials . the required sample size at a = 0.05 , a power of 0.85 and an effect size of 0.6 , was a priori computed using g power v.3.1.5 ( franz ful , universitat kiel , germany ) . visually intact teeth were carefully selected from recently extracted teeth , without the presence of hypomineralized or hypolplastic areas , caries or fracture lines on their buccal or lingual surfaces . in order to prepare cavities , as much as possible on the same horizontal tooth plane , the roots of all teeth , 2 - 3 mm below their cemento - enamel junction , were cut and removed . two buccal ans two linguall cavities of 2.5 mm in depth were prepared in each tooth under air - water spray , with a diamond bur rotating in a high - speed hand piece . two of the cavities were opened with a 0.8 mm in diameter bur ( fgd0 , strauss and co ) and two with a 1.0 mm diameter ( fgd1 , strauss and co ) . cavity depth was established with a length mark on each bur and cutting direction was facilitated by guiding the tooth against a stabilized hand piece with a horizontally rotating bur [ figure 1 ] . no special attention was given for opening the cavities with an exact width or depth , in order to have a range of cavities with differences in width or depth and facilitate their correlation with restorations gaps and voids . only four cavities were allowed to be opened by the same bur and cavities of different diameter were distributed equally to labial and lingual surfaces . after preparation of the cavities , all teeth were stored in water of 37c until their first microtomographic examination by skyscan 1072 microtomographer ( micro xct , model 1072 , skyscan aartselaar , belgium ) . the cavities were examined before insertion of the filling material , to calculate the precise cavity dimensions and assist the estimation of gaps and voids in the restored cavities at the second microtomographic examination [ figure 2 ] . operating parameters used for skyscan 1072 microtomographer upper left : microtomographic section of a tooth showing the prepared cavities . lower : a screen image showing the binarization settings with tview software , before calculation starts . the restorative materials used to fill the experimental cavities are shown in table 2 , and the procedure followed for each material is described below . the assignment of restorative material and bur diameter on tooth surfaces was based on a latin square design , in order to have teeth , with all the designed treatment variables , in a systematic rather than a random fashion . all cavities were acid etched with 37% ortho - phosphoric acid for 30 s , rinsed with water for 2 - 3 s and dried with air for 4 - 5 s and 1 - 2 absorbent paper points . cavities assigned to receive composite resins were lined carefully with adhesive according to manufacturer instructions ( prime and bond nt , dentsply int inc , milford , usa ) and polymerized for 20 s , using a curing unit with a light intensity of 550 - 600 mw / cm ( demetron lc / kerr gmbh , rastatt , germany ) . it must be mentioned that adhesive material was used with caution since excess of adhesive remain undetected by x - rays and even a small excess could be measured as gap . materials used in the study aeliteflo / bisco inc , chicago , usa ( af ) was placed in the cavity with a small diameter tip on material syringes . the tip was initially placed at the bottom of the cavity and was gradually withdrawn in an outward direction as the material was injected into the cavity . the material was polymerized for 30 s , and the excess , was finished and polished using medium , fine and superfine sof - lex discs ( 3 m espe , dental products , st . permaflo / ultradent products inc , stlouis / usa(pf ) was placed in the cavity exactly as af . aelite aesthetic / bisco inc , chicago , usa ( aa ) was placed in the cavity in small parts and condensed with the use of a dentin excavator , modified to a small diameter condenser ( 0.8 mm ) , in order to enter the cavities . after condensation and polymerization , the same finishing and polishing procedure as in af was followed . fuji ix / gc corporation , tokyo , japan ( fu ) was inserted in the cavity after activation of the capsule according to manufacturer 's instructions , by placing the capsule tip at the entrance of the cavity and condensing the material with the use of the same condenser , as before . immediately after finishing and polishing of restorations , teeth were placed in distilled water of 37c and retrieved after 24 h to receive 500 thermal cycles ( 5 - 55c , 3 min each ) . the second x - ray microtomographic examination was made under the same operating parameters with the first examination , immediately after thermocycling procedure . the volume amount of empty spaces within the material or between material and cavity walls was calculated on this second series of digital sections , using the tview v.1.3 software ( skyscan n.v . the software calculates on binary images of sample 's tomographic sections , the different in density areas , within a predefined region of interest ( roi ) . running all relative sections , the total volume ( vt ) and the volume fraction percent ( vf % ) of the interested densities ( called active pixels ) in the roi can be calculated . a specific process had to be followed , to ensure that all calculations were based exactly on the same roi . aartelaar , belgium , the number of horizontal sections covering the width of the cavity was determined . the sections showing all four cavities at their largest width were located and copied on a photoshop cs file . a circular area over the cavity was drawn on a transparent layer with the marquee tool . then , the circular marquee line was copied in new transparent layers , moved and applied to all four cavities for an exact positioning and all four layers were combined and saved as a single .bmp file . this process was repeated for all tooth cavities [ figure 2 ] and the saved .bmp files were placed as the last tomographic image , in order to remain available , but outside the range of the selected sections . the inserted image in each set of tooth microtomographs was located and the roi was drawn over the selected areas . the upper and lower sections of each cavity were inserted , and the color palette was set to binary colors ( 247 in level-1 and 255 in level-2 ) , for best discrimination of empty spaces from tooth / material substance [ figure 2 ] . the three - dimensional analysis was used , in order to collect information from the set of predefined horizontal sections on the variables described below . this three - dimensional analysis was repeated three times for each cavity and for all teeth . data collected concerned the following morphometric values : vt , the total selected volume throughout all the cross sections , vf % , the volume percent of the selected item ( empty space ) in an area , st , the total surface area of the item . the interest was centered on vf % and its relation to the initial volume of cavity dimensions and restorative material type . vt , the total selected volume throughout all the cross sections , vf % , the volume percent of the selected item ( empty space ) in an area , st , the total surface area of the item . the interest was centered on vf % and its relation to the initial volume of cavity dimensions and restorative material type . aartelaar , belgium , the number of horizontal sections covering the width of the cavity was determined . the sections showing all four cavities at their largest width were located and copied on a photoshop cs file . a circular area over the cavity was drawn on a transparent layer with the marquee tool . then , the circular marquee line was copied in new transparent layers , moved and applied to all four cavities for an exact positioning and all four layers were combined and saved as a single .bmp file . this process was repeated for all tooth cavities [ figure 2 ] and the saved .bmp files were placed as the last tomographic image , in order to remain available , but outside the range of the selected sections . the inserted image in each set of tooth microtomographs was located and the roi was drawn over the selected areas . the upper and lower sections of each cavity were inserted , and the color palette was set to binary colors ( 247 in level-1 and 255 in level-2 ) , for best discrimination of empty spaces from tooth / material substance [ figure 2 ] . the three - dimensional analysis was used , in order to collect information from the set of predefined horizontal sections on the variables described below . this three - dimensional analysis was repeated three times for each cavity and for all teeth . data collected concerned the following morphometric values : vt , the total selected volume throughout all the cross sections , vf % , the volume percent of the selected item ( empty space ) in an area , st , the total surface area of the item . the interest was centered on vf % and its relation to the initial volume of cavity dimensions and restorative material type . vt , the total selected volume throughout all the cross sections , vf % , the volume percent of the selected item ( empty space ) in an area , st , the total surface area of the item . the interest was centered on vf % and its relation to the initial volume of cavity dimensions and restorative material type . the location of voids and gaps was evaluated using the tview software and its no1 color palette . all relative to each cavity .bmp images ( 1024 1024 pixels ) were viewed by two examiners for the location of voids if present , on a 19 computer screen . for their classification three locations were recorded : the material itself , the side walls of the cavity andthe bottom of the cavity . voids at the walls or bottom of the cavity were actually the gaps at the material / cavity interface . for each cavity , one , two or all three locations were recorded , always based on the agreement of both examiners . voids at the walls or bottom of the cavity were actually the gaps at the material / cavity interface . for each cavity , one , two or all three locations were recorded , always based on the agreement of both examiners . kolmogorov - smirnov tests for normality and bartlett 's test for the equality of variances indicated the use of kruskal - wallis nonparametric test to analyze the data for differences between materials , in respect to the morphometric parameters . mann - whitney test was also used to search for possible differences between low and high viscosity materials , for each morphometric parameter . the degree of correlation of cavity dimensions with all morphometric parameters was analyzed by spearman 's test . all statistical tests performed with medcalc v.10.0 ( medcalc software , mariakerke , belgium ) at a 0.05 level of significance . unfilled cavity dimensions were measured and found to have a mean length of 2.6 0.4 mm and a mean width 1.30 0.19 mm . the mean values of all morphometric elements ( vt , vf and st ) , estimated by three - dimensional analysis for the cavities filled with four restorative materials are shown in table 3 . morphometric parameters of voids and gaps upper left : a tooth section showing two of the filled cavities with voids within fuji ix ( fu ) material . lower right : voids at the bottom of an fu and in the middle of an aa restoration . statistical analysis by kruskal - wallis nonparametric analysis of variance indicated significant differences among filled cavity groups , for all three parameters ( kwvt = 38.0 , kwvf = 38.29 , kwst = 29.28 , p < 0.0001 ) . post - hoc analysis based on pairwise comparisons showed significantly higher values of vf % in cavities filled with fu than those filled with other materials ( p < 0.05 ) [ table 3 ] . restorations with af and pf materials showed the lowest vf % values of all ( p < 0.05 ) . mann - whitney test also indicated a significant difference between cavities filled with high and low viscosity materials ( u = 565.5 , p < 0.0001 ) . cavity length , width and volume of high and low viscosity materials as well as in most individual materials correlated poorly and not significantly with vf % . however , in cavities filled with aa , a significant correlation was found between vf % and depth of the cavity . spearman 's rho coefficients of vf % with cavity dimensions the proportion of cavities with voids at different locations ( within the material , bottom and side walls of the cavity ) is shown in table 5 . in 50 - 75% of the restorations , voids were present within the material , without difference in their proportion among the different materials . in 90 - 100% of the cavities filled with aa and fu , gaps were present at the bottom of the cavity , and significantly higher than those filled with af or pf . all cavities filled with aa showed also gaps at the side walls of the cavity , significantly higher than all other materials . lower and upper limits of the 95% ci for the proportion of cavities with voids within the material , and gaps at the bottom or side walls of the cavity the results showed that the procedure followed to compare voids fraction among different cavities on the same sample , is capable of revealing significant differences . the study rejected all three hypotheses : for no differences between materials in vf % of voids ( and gaps),for no association of cavity dimensions with vf % of voids ( and gaps ) , andfor no association of voids location with a specific restorative material . for no differences between materials in vf % of voids ( and gaps ) , for no association of cavity dimensions with vf % of voids ( and gaps ) , and for no association of voids location with a specific restorative material . the procedure introduced in this study for the selection of exactly the same roi on all cavities was not a simple one , but since differences between repeated measurements ranged only from 0.002 to 0.005 vf % , this indicates that the method was able to reveal small differences in the volume of voids in very conservative cavities . however , the need to add extra digital sections to a number of cavities smaller in depth than others , or the inclusion in the experiment of materials with high variability in the volume of voids , like fu material , may introduce an error to the measurements . in our study , the low viscous materials were the best among different materials in filling the ultraconservative cavities without gap and this is in agreement with the results of other studies for normal cavities . found that thinner composites had fewer problems with voids and wall adaptation than thicker materials . peutzfeldt and asmussen , who also studied gap formation , found a positive correlation of high viscosity and polymerization shrinkage with gap formation . moreira da silva et al . found a correlation of gap and voids formation with high viscous flow and low flexural modulus of composite materials . these results can be explained by the high thixotropic effect of low viscous composite materials , and their low polymerization shrinkage due to their low modulus of elasticity . opdam et al . showed that injection technique , like the one we used with low viscous composites , leads to reduced voids formation within the material and a smaller interfacial gaps . hence , in cavities of very small diameter ( 1.0 - 1.5 mm ) where the insertion of the restorative material and its adaptation to the cavity walls is difficult , low viscous material should be syringed deep into the cavity instead of using higher viscosity composites . thicker composite materials even if they are managed to be inserted in such cavities can potentially form internal voids between layers , and for this reason their use should be done with caution . glass - ionomer material was inserted with difficulty in small cavities , due to its adherence to the instruments and the cavity walls during insertion . as a result of this this type of material should not be used in such small cavities unless perhaps a special small in diameter tip designed to allow proper insertion of the material deep into the cavity before its hardening . our study also showed a significant correlation of initial cavity volume with vf of spaces around and within aa filling material only . this positive correlation of deeper or wider cavities with the presence of voids is probably a result of higher polymerization contraction in larger restorations , as he et al . their study showed that in larger cavities with a high c - factor ( class i and v ) , the bonding was riskier than in small cavities . therefore , larger cavities filled with high viscous composite material may present a higher amount of voids and gaps . our study showed that the voids within the restorative material were not associated with a specific type of material . however , gaps at the bottom of the cavity were more frequently associated with the more viscous aa and fu materials , as other studies indicated . they presented a higher polymerization contraction than af and pf materials , and need a higher force and more attention to make a closer contact with the bottom of the cavity . the reason is probably that polymerization contraction , which is evident with all cavity walls in a nonbonded restoration , creates a space at the sides of the cavity , more evidently than in other materials . fu , for instance , has the ability of creating a bond with the side walls of the cavity , but also seems to create larger gaps at the bottom of the cavity , which helps to accommodate a significant part of its contraction during hardening . it must be noticed that pixel size in microtomographic images was 14 in this study , although system 's detection limit was 1.8 . for this reason , smaller in size voids could not be recorded or located and voids of such dimensions may exist in all types of materials and locations . this is probably the limitation of the suggested method and the reason why low viscous materials show a low proportion of voids or gaps . however , newer ct technology ( x - ray computed nanotomography ) will be able to estimate gaps and voids more accurately . voids are present in the same frequency within the mass of all restorative materials , but gaps are present more frequently in restorative materials with high viscosity , at the bottom and the side cavity walls . for these reasons , more research is needed to refine the available techniques for placing correctly and precisely a restorative material within a very conservative cavity , the way of using adhesives in cavities of a very small opening , the type of material that can actually minimize the hardening shrinkage of the material and of course the measurement of voids of very small dimensions . there were differences between restorative materials in the vf % of voids and gaps remaining after condensation of the materials in the cavities.low viscous composite resins were the best in filling cavities without voids or gaps.high viscous composite and glass - ionomer materials produced the highest amount of internal voids and gaps.cavity depth , width and volume do correlate with the amount of voids and gap spaces , but only for the high viscous composite material.voids are located in the same frequency within all materials , but gaps are more frequently located within high viscous composites , both at the bottom and the side cavity walls . there were differences between restorative materials in the vf % of voids and gaps remaining after condensation of the materials in the cavities . high viscous composite and glass - ionomer materials produced the highest amount of internal voids and gaps . cavity depth , width and volume do correlate with the amount of voids and gap spaces , but only for the high viscous composite material . voids are located in the same frequency within all materials , but gaps are more frequently located within high viscous composites , both at the bottom and the side cavity walls . the authors of this manuscript declare that they have no conflicts of interest , real or perceived , financial or non - financial in this article . the authors of this manuscript declare that they have no conflicts of interest , real or perceived , financial or non - financial in this article .

background : volume fraction ( vf ) and location of internal voids and gaps in relation to material type and cavity dimensions in ultraconservative restorations were investigated in this study.materials and methods : forty - eight round cavities of 1.3 mm mean diameter and 2.6 mm mean depth were made on buccal and lingual surfaces of recently extracted human teeth . these were filled and thermocycled with two low viscosity composites ( aeliteflo lv [ af ] , permaflo [ pf ] ) , one high viscosity composite ( aelite aesthetic enamel [ aa ] ) and one glass - ionomer ( gcfuji ix gp ) . x - ray microtomography , following a specific procedure , was applied to all cavities before and after their restoration , using skyscan-1072 microtomographer . vf percent ( vf% ) and location of voids and gaps were recorded and analysed statistically at a = 0.05 . kruskal - wallis nonparametric analysis of variance , post - hoc analysis , mann - whitney test , spearman 's correlation analysis were used to analyze data.results:cavities filled with af and pf showed significantly lower vf % of voids and gaps than all other restorations ( p < 0.05 ) . only for the cavities filled with aa , cavity width and depth was significantly correlated with vf % ( p < 0.05 ) . 50 - 75% of the filled cavities contained internal voids regardless of the restorative material ( p > 0.05 ) . the proportion of cavities with gaps at the bottom and side walls was lower in those filled with af and pf ( p < 0.05).conclusion : cavities filled with low viscosity composites presented the lowest amount of internal voids and gaps . glass - ionomer and high viscosity composite restorative materials showed the highest amount of interfacial gaps . only in the high viscosity composite restorations the amount of voids and gaps correlated with the cavity depth , width and volume .

INTRODUCTION MATERIALS AND METHODS Sampling Cavity preparations First microtomographic examination Cavity filling Second microtomographic examination Selection of measuring areas Measuring process Voids and gaps location Statistical analysis RESULTS DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

PMC32187

the homeotic / hox genes encode a network of evolutionarily conserved homeodomain transcription factors that are involved in the specification of segmental identity along the anterior - posterior body axis of animals as diverse as insects and vertebrates [ 1 , 2 , 3 , 4 , 5 , 6 ] . in drosophila , these genes are arranged on the chromosome in two gene clusters known as the antennapedia and bithorax complexes . there is a correlation between the relative position of the hox genes within the cluster and their spatial and temporal expression pattern in the body in that genes located towards the 3 ' end are expressed more anterior and earlier than genes located towards the 5 ' end ( spatial and temporal colinearity ) [ 7 , 8 , 9 , 10 , 11 ] . given their central role in developmental processes , it has been proposed that the homeoproteins do not act directly to specify morphological differences but rather control a battery of subordinate genes encoding cellular functions directly required in differentiation [ 12 , 13 ] . in search of these subordinate genes , various strategies such as enhancer trapping , immunoprecipitation of chromatin fragments , subtractive hybridization , selection for binding sites in yeast , and heat - shock - induced overexpression have been used [ 9 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21].only a small number of target genes of homeoproteins have been identified to date , however ; most of these encode either transcription factors or cell - signaling molecules . in contrast to these results , recent studies suggest that homeoproteins may bind at significant levels to the majority of genes in the drosophila embryo and regulate a large number of downstream genes [ 22 , 23 ] . here it is the most proximal gene within the drosophila antennapedia complex ; it encodes an antennapedia - like q50 homeodomain transcription factor and is one of the most anteriorly expressed homeotic genes along the anterior - posterior body axis [ 24 , 25 , 26 , 27 ] . genetic studies have demonstrated that lab is required for proper head formation and for the specification of cellular identity in the midgut as well as in the embryonic brain . the lab gene and its vertebrate hox1 orthologs are among the best - characterized examples of evolutionary conservation of structure , expression and function of hox genes in animal development [ 31 , 32 , 33 , 34 , 35 ] . to address the question of which and how many downstream genes are under control of lab , we used a combination of in vivo overexpression techniques and quantitative transcript imaging with oligonucleotide arrays . by using transgenic flies carrying the lab gene under the control of a heat - inducible promoter , we ubiquitously overexpressed lab following heat - shock treatment in drosophila embryos . we then used high - density oligonucleotide arrays representing 1,513 identified drosophila genes for large - scale detection and quantification of induced gene expression [ 36 , 37 , 38 , 39 ] . quantitative reverse - transcriptase pcr on a selection of these genes verified the differential expression levels in response to heat - shock - induced overexpression of lab . our findings identify a number of novel candidate downstream genes for lab and thus show that oligonucleotide arrays are powerful tools for analyzing , at a genome - wide level , the number , identity and quantitative expression level of genes in the drosophila embryo . in this study , transgenic fly strains carrying the lab coding sequence under control of the heat - inducible hsp70 promotor were used . stage 10 - 17 embryos were given a 25 minute heat pulse to overexpress lab , and allowed to recover for 25 minutes ( see materials and methods for heat - shock protocol ) . ubiquitous overexpression of lab was verified by whole mount in situ hybridization with a lab - specific antisense rna probe . ubiquitous overexpression of labial protein ( lab ) was verified by immunocytochemistry with an anti - lab antibody . these experiments demonstrated that both lab rna and lab protein were strongly overexpressed 50 minutes after the onset of heat shock in these strains ( figure 1 ) . wild - type control flies were subjected to an identical heat - shock regime . following ubiquitous overexpression of lab , transcript profiles were analyzed using a high - density oligonucleotide array and compared to the transcript profiles of heat - shocked wild - type control embryos . for each of the two experimental conditions ( ' hs - wt'and ' hs - lab ' ) , four replicate experiments were performed and the data set was analyzed with an unpaired t - test ( see and materials and methods ) . the genes represented on the oligonucleotide array correspond to probe sets that are complementary to 1,513 identified and sequenced drosophila genes . most of these genes can be grouped into 14 functional categories according to the nature of the encoded protein . at a significance level of p 0.01 , a total of 96 genes were found to be differentially regulated following lab overexpression compared with heat - shocked wild - type control embryos . this corresponds to 6.3% of the genes represented on the array . at a significance level of p 0.05 , 205 genes were found to be differentially regulated following lab overexpression compared with heat - shocked wild - type control embryos ( data not shown ) . the relative distribution of lab - regulated genes in particular functional classes , as well as the percentage of genes regulated within a given functional class , were comparable between the p 0.01 group and the p 0.05 group . only genes that were differentially expressed at a significance level of p 0.01 are considered further . we propose these genes to be potential direct or indirect downstream targets for the homeodomain transcription factor labial . when ubiquitously expressed in the embryo , lab caused a significant transcriptional response among a wide variety of genes belonging to all functional classes represented on the array ( table 1 ) . the functional class with the highest absolute number of differentially regulated genes was ' transcriptional regulation ' ( n = 20 ) . other functional classes with high numbers of differentially regulated genes were ' metabolism ' ( n = 13 ) , ' proteolytic systems / apoptosis ' ( n = 12 ) , ' cell - surface receptors / cell adhesion molecules ( cams)/ion channels ' ( n = 12 ) , and ' rna binding ' ( n = 7 ) . relative to the number of genes represented on the array within a given functional class , the highest relative percentage of differentially regulated genes was found in the functional classes ' proteolytic systems / apoptosis ' ( 19.4% ) , ' cell cycle ' ( 13.5% ) , ' transposable elements ' ( 11.4% ) , ' chromatin structure ' ( 11.1% ) , ' rna binding ' ( 11.9% ) , and ' transcriptional regulation ' ( 7.6% ) . figure 2 shows the lab - regulated genes and presents a quantitative representation of the change in expression levels for these genes . of the 96 genes that were differentially regulated , 48 showed increased expression levels and 48 showed decreased expression levels the gene with the highest increase in expression level ( 26-fold ) was lab itself , in accordance with our experimental procedure . increases in expression levels above 10-fold were also observed for bicaudal c ( bicc ) , swallow ( swa ) and oskar ( osk ) , all encoding proteins involved in rna binding , as well as for the wings apart - like ( wapl ) gene belonging to the functional class ' chromatin structure ' . the increased expression levels in bicc , swa , and osk are surprising , as all these genes are known to function as maternal control genes during early embryogenesis [ 41 , 42 ] . as lab activity is normally only observed from gastrulation onwards , this suggests that high levels of widespread ectopic lab expression are able to activate genes which under wild - type conditions show spatio - temporal expression domains that do not overlap with that of lab . one encodes the enzyme ubiquitin carboxy - terminal hydrolase , whose mammalian homolog has also been found to be differentially upregulated by ectopic overexpression of the lab ortholog hoxa1 . increased expression levels in the 1.5 - 5-fold range were prominent in several functional classes . for example , in the functional class ' proteolytic systems / apoptosis ' , 12 of 13 differentially regulated genes were upregulated and most of these showed increased expression levels ranging between 1.5 and 5 . strikingly , in the functional classes ' cell cycle ' and ' transcription / replication / repair ' all the differentially regulated genes were upregulated . thus , differentially expressed genes such as twine ( twe ) , cyclin b ( cycb ) and cyclin d ( cycd ) , belonging to the functional category ' cell cycle ' , were all upregulated following lab overexpression . it is notable in this respect that recent experiments carried out on mammalian cell lines showed that ectopic overexpression of the lab ortholog hoxa1 also causes differential upregulation of cell - cycle regulatory proteins . decreases in expression levels in the 10-fold and above range were not observed , and decreases in the 5 - 10-fold range were only seen for the transposable r2 rdna element gene . decreased expression levels in the 1.5 - 5-fold range were , however , prominent in the functional class ' transcriptional regulation ' and in the functional class ' cell - surface receptors / cams / ion channels ' . thus , almost three - quarters of the differentially regulated genes encoding transcription factors showed significant decreases in expression levels following lab overexpression . for example , the genes prospero ( pros ) , distal - less ( dll ) , tailup / islet ( tup ) , mirror ( mirr ) , huckebein ( hkb ) and abrupt ( ab ) were all downregulated . interestingly , it has been shown that distal - less is a direct target of homeotic gene control , and recent genetic studies demonstrated that tailup / islet expression in the lab - specific territory of the embryonic drosophila brain is dependent on lab gene action . as with the functional class ' transcriptional regulation ' , 10 out of 12 genes representing the functional category ' cell - surface receptors / cams / ion channels ' were downregulated , including the genes derailed ( drl ) , frizzled 2 ( fz2 ) , neurotactin ( nrt ) , neurexin ( nrx ) , rhomboid ( rho ) and 18 wheeler ( 18w ) . as is the case for tailup / islet , neurotactin expression in the lab - specific territory of the embryonic drosophila brain is dependent on lab gene action . the 18w locus has been identified as a binding site of the homeotic protein ubx in polytene chromosomes . to verify the differences in gene expression level after heat - shock - induced overexpression of lab as compared to heat - shocked wild - type embryos , quantitative rt - pcr was performed on selected candidate target genes . changes in expression levels were determined for eight genes that were differentially regulated following lab overexpression , namely lab , swa , ubiquitin conjugating enzyme 4 ( ubcd4 ) , twe , cycb , ubiquitin carboxy - terminal hydrolase ( uch ) , scratch ( scrt ) and phosphoenolpyruvate carboxykinase ( pepck ) . the gene squid ( sqd ) , whose expression level remained unchanged under both experimental conditions , served as a control . as indicated in table 2 , these experiments showed that the changes in relative expression level , as measured by rt - pcr , are consistent with the data obtained with the oligonucleotide arrays . we have used a novel combination of manipulative genetics and functional genomics to gain further insight into homeotic gene action in drosophila from a genomic perspective . using inducible overexpression and quantitative transcript imaging through oligonucleotide arrays , we have identified 96 genes ( only 6.3% of the 1,513 identified genes represented on the oligonucleotide array ) whose expression levels change significantly following lab overexpression . these findings suggest that lab regulates a limited and distinct set of candidate downstream genes . this appears to contrast with previous reports indicating that in late embryogenesis the majority of drosophila genes are under control of homeoproteins [ 23 , 44 ] . it should be stressed , however , that a number of features of our functional genomic analysis prevent a direct comparison with these reports , which are based on dna - binding studies . first , although our analysis can quantify gene expression accurately and simultaneously for many identified genes , the temporal and spatial resolution of our analysis is low . this is because our experimental design averages gene expression throughout the embryo and during several embryonic stages . in consequence , our analysis may fail to detect genes that are only expressed in a small subset of cells or during a very restricted time period in embryogenesis . second , our overexpression protocol makes it difficult to control the level of lab protein as well as the temporal dynamics and stability of this protein . as different levels of a given homeoprotein can have different functional consequences in terms of developmental specificity [ 29 , 45 ] , the high level of lab protein may bias the set of candidate downstream target genes identified . third , in our studies lab overexpression is not accompanied by concomitant overexpression of cofactors , which are thought to act together with homeotic proteins to determine their in vivo target specificity [ 34 , 46 ] . it should be noted that the gene mirror , which has been proposed to be an additional cofactor for homeoprotein specificity , was detected as downregulated following lab overexpression . although the question of the total number of target genes that are regulated by homeoproteins in vivo must await further analysis , our genomic perspective of lab gene targets does reveal several specific features of homeoprotein action . first , our results demonstrate that the homeodomain transcription factor lab acts on numerous candidate target genes that also encode transcription factors . the category ' transcriptional regulation ' comprises one of the largest sets of differentially regulated genes following lab overexpression . this is consistent with the idea that homeobox genes establish developmental patterns by acting through a cascade of transcription factors which regulate the expression of their own subset of downstream genes [ 1 , 2 , 9 , 15 ] . second , our data indicate that upregulation of gene expression is prominent in several functional classes . thus , virtually all of the lab - regulated genes in the functional classes ' cell cycle ' , ' transcription / replication / repair ' , and ' proteolytic systems / apoptosis ' show increased expression . third , our results show that lab overexpression causes not only widespread activation but also widespread repression of gene expression . thus , of the 96 genes that are potential targets of lab , half are downregulated by overexpression of this homeobox gene . this widespread repression is especially pronounced in the functional classes of ' transcriptional regulation ' and ' cell - surface receptors / cams / ion channels ' . for example , following lab overexpression , over 80% of the differentially regulated genes encoding cell - surface receptors / cams / ion channels showed decreased expression . taken together , our results identify a large number of novel candidate downstream genes of the homeodomain transcription factor lab . to our knowledge , most of these 96 identified and sequenced genes have not been previously shown to be lab targets . at present , we do not know which genes are direct targets ( regulated directly by lab protein binding to dna regulatory sequences ) or indirect targets of lab gene action . furthermore , our results demonstrate that oligonucleotide arrays are useful tools for analyzing , at a genome - wide level , the number , identity and quantitative expression levels of candidate downstream genes differentially regulated in vivo by developmental control genes . this confirms the general utility of microarrays for studying diverse molecular and cellular processes in drosophila [ 48 , 49 , 50 ] . considering the evolutionary conservation of gene structure , expression and function [ 1 , 35 ] , we propose that these results obtained in drosophila will also be valid for lab orthologs in other animals , including vertebrates . it will now be important to determine which of the detected candidate downstream genes in drosophila are direct targets and how they exert the developmental genetic programs imposed by lab gene action . overexpression of lab , we used the line p(whs - lab ) with a heat - shock lab construct homozygous on the x chromosome . all fly stocks were kept on standard cornmeal / yeast / agar medium at 25c . embryos were collected overnight for 12 h on grape juice plates , further kept for 4 h at 25c and then subjected to a 36c heat shock for 25 min , followed by a recovery period of 25 min at 25c before rna isolation . therefore , at the time of rna isolation these embryos were at embryonic stages 10 - 17 ( stages according to ) . embryos younger than embryonic stage 10 were not used , as heat shock in these earlier stages results in lethality . for in situ hybridization , digoxigenin - labeled sense and antisense lab rna probes were generated in vitro , with a dig labeling kit ( roche diagnostics ) and hybridized to whole - mount embryos following standard procedures . hybridized transcripts were detected with an alkaline phosphatase - conjugated anti - digoxigenin fab fragment ( roche diagnostics ) using nitro blue tetrazolium ( nbt ) and 5-bromo-4-chloro-3-indolyl phosphate ( bcip ) ( sigma ) as chromogenic substrates . the histochemical staining was performed using the vectastain elite abc kit ( vector laboratories ) . embryos were mounted in canada balsam ( serva ) and photographed with a prog / res/3008 digital camera ( kontron electronic ) on a zeiss axioskop microscope with differential interference contrast optics . gene expression analysis was performed as described , using a custom - designed drosophila oligonucleotide array ( roez003a ; affymetrix ) . the genes represented on the array and considered in this study correspond to 1,513 sequenced drosophila genes encoding open reading frames deposited in swiss - prot / trembl databases as of spring 1998 . for a complete list of these genes each gene is represented on the array by a set of 20 oligonucleotide probes ( 25mers ) matching the gene sequence . to control the specificity of hybridization , the same set of probes , containing a single nucleotide mismatch in a central position , is represented on the array . the difference between the perfect match hybridization signal and the mismatch signal is proportional to the abundance of a given transcript and calculated as its average difference value ( avg diff ) . drosophila genes that were not unambiguously represented by a probe set of 20 probe pairs on the array were excluded from further analysis ( 29 probe sets were not used in this study ) . initial experiments designed to determine the sensitivity and reproducibility of hybridization showed that the use of total rna versus poly(a ) rna as a template for cdna synthesis and subsequent amplification ( synthesis of crna ) gave comparable results , despite the fact that we consistently detected 5s rna and histone genes present on the array with crna derived from total rna . on the basis of these findings , all experiments were carried out using a total rna protocol . total rna was isolated from 200 mg of embryonic tissue , using guanidinium isothiocyanate in combination with acidic phenol ( ph 4.0 ) ( fast rna tube green kit from bio101 ) in a fast prep homogenizer fp120 ( bio 101 ) . after precipitation , the rna was dissolved in depc - treated water ( ambion ) and spectrophotometrically quantified using a genequant rna / dna calculator ( pharmacia biotech ) . cdna was synthesized upon total rna as a template , using the superscript choice system for cdna synthesis ( gibco / brl ) with a t7-(t)24 dna primer . this primer ( 5'-ggccagtgaattgtaatacgactcactatagggaggcgg-(t)24vn-3 ' ) was purified by page . for first - strand cdna synthesis , a typical 40 l reaction contained 25 g rna , 200 pmol t7-(t)24 primer , 500 m of each dntp and 800 units reverse transcriptase ( amv superscript ii ) . second - strand cdna synthesis was carried out at 18c for 2 h in a total volume of 340 l , using 20 units escherichia coli dna ligase , 80 units e. coli dna polymerase i and 4 units rnase h in the presence of 250 m of each dntp . after second - strand cdna synthesis , 0.5 l rnase a ( 100 mg / ml ) ( qiagen ) was added and the samples were incubated at 37c for 30 min . thereafter , 7.5 l proteinase k ( 10 mg / ml ) ( sigma ) was added and the samples were further incubated at 37c for another 30 min . after cdna synthesis was completed , samples were phenol - chloroform extracted , using phase lock gel ( 5 prime-3 prime ) and ethanol precipitated . biotinylated antisense crna was synthesized from the dsdna template , using t7 rna polymerase ( megascript t7 kit : ambion . ) . a 20 l reaction volume contained between 0.3 - 1.5 g cdna , 7.5 mm of both atp and gtp , 5.6 mm of both utp and ctp and 1.8 mm of both biotinylated bio-16-utp and bio-11-ctp ( enzo diagnostics ) and 2 l 10x t7 enzyme mix . the reaction was incubated at 37c for 8 h. thereafter , the unincorporated ntps were removed by running the sample over an rneasy spin column ( qiagen ) . samples were precipitated , taken up in 20 l depc - treated water and spectrophotometrically quantified . thereafter , 40 g of the biotinylated antisense crna was fragmented by heating the sample to 95c for 35 min in a volume of 25 l , containing 40 mm tris - acetate ( ph 8.1 ) , 100 mm potassium acetate , 30 mm magnesium acetate . after the fragmentation gene chips were pre - hybridized with 220 l hybridization buffer ( 1x mes ( ph 6.7 ) , 1 m nacl , 0.01% triton , 0.5 g/l acetylated bsa , 0.5 g/l sonicated herring sperm dna ) for 15 min at 45c on a rotisserie ( heidolph ) at 60 rpm . hybridization was done in a final volume of 220 l hybridization buffer , containing 40 g fragmented biotinylated crna . hybridizations were carried out for 16 h at 45c with mixing on a rotisserie at 60 rpm . after hybridization , the arrays were briefly rinsed with 6x sspe - t ( 0.9 m nacl , 0.06 m nah2po4 , 6 mm edta , 0.01% triton ) and washed on a fluidics station ( affymetrix ) . hybridized arrays were stained with 220 l detection solution ( 1x mes buffer , containing 2.5 l streptavidin - r phycoerythrin conjugate ( 1 mg / ml ) ( molecular probes ) ) and 2.0 mg / ml acetylated bsa ( sigma ) at 40c for 15 min and washed again . pixel intensities were measured with a commercial confocal laser scanner ( hewlett packard ) and expression signals were analyzed with commercial software ( genechip 3.1 ; affymetrix ) . detailed data analysis was carried out using race - a ( roche ) , access 97 and excel 97 ( microsoft ) software . for quantification of relative transcript abundance the normalized average difference value ( avg diff ) was used . for each of the three experimental conditions ( wt , hs - wt , hs - lab ) , four replicates were carried out ( for the experimental conditions wt and hs - wt see , including the supplementary data ) . for the difference of the means of the avg diff values over the four replicates between condition 1 ( hs - wt ) and condition 2 ( hs - lab ) , a t - test was performed . moreover , for downregulation , the mean avg diff value of a gene had to be above or equal to 50 in condition 1 ; for upregulation , the mean avg diff value of a gene had to be above or equal to 50 in condition 2 . genes which had a normalized avg diff below 20 obtained automatically an avg diff of 20 ( race - a protocol ) . to obtain a comprehensive analysis of the number and identity of genes differentially regulated by lab , candidates that were already differentially expressed in heat - shocked wild - type embryos compared to non - heat - shocked wild - type controls , were excluded from further analysis ( and data not shown ) . previously , we have used quantiative rt - pcr to confirm that relative expression level changes in the 1.5-fold and above range , as detected on this array , accurately reflect differences in mrna abundance in vivo in drosophila embryos . in consequence , in this report only relative expression level changes in the 1.5-fold and above range are presented . three hundred nanograms of poly(a ) rna , isolated from heat - shocked wild - type embryos and heat - shocked hs - lab embryos ( mrna isolation kit ; roche diagnostics ) , was reverse transcribed with amv - rt and random hexamers ( first - strand cdna synthesis kit for rt - pcr ; roche diagnostics ) . pcr was performed with 100 pg of template dna and gene - specific primers ( designed , using seq web , wisconsin package version 10.0 ; gcg ) on a light cycler ( lightcycler ; roche diagnostics ) . continuous fluorescence observation of amplifying dna was done using sybr green i ( lightcycler - faststart dna master sybr greeni ; roche diagnostics ) . after cycling , a melting curve was produced by slow denaturation of the pcr end - products to check the specificity of amplification . to compare the relative amounts of pcr products , we monitored the amplification profile on a graph , displaying the log of the fluorescence against the number of cycles . relative fold changes for a given gene under both conditions ( heat shock wt versus heat shock hs - lab ) were calculated using the fit point method ( lightcycler operator 's manual , version 3.0 ; roche diagnostics ) . the genes represented on the high - density oligonucleotide array were grouped into 14 functional classes according to the function of the gene product and currently available genetic data . for this , notations in flybase , interactive fly and swiss - prot / trembl databases were used . a comprehensive presentation of all the genes represented on the oligonucleotide array as well as their attribution to functional classes is given as supplementary data to . overexpression of lab , we used the line p(whs - lab ) with a heat - shock lab construct homozygous on the x chromosome . all fly stocks were kept on standard cornmeal / yeast / agar medium at 25c . embryos were collected overnight for 12 h on grape juice plates , further kept for 4 h at 25c and then subjected to a 36c heat shock for 25 min , followed by a recovery period of 25 min at 25c before rna isolation . therefore , at the time of rna isolation these embryos were at embryonic stages 10 - 17 ( stages according to ) . embryos younger than embryonic stage 10 were not used , as heat shock in these earlier stages results in lethality . for in situ hybridization , digoxigenin - labeled sense and antisense lab rna probes were generated in vitro , with a dig labeling kit ( roche diagnostics ) and hybridized to whole - mount embryos following standard procedures . hybridized transcripts were detected with an alkaline phosphatase - conjugated anti - digoxigenin fab fragment ( roche diagnostics ) using nitro blue tetrazolium ( nbt ) and 5-bromo-4-chloro-3-indolyl phosphate ( bcip ) ( sigma ) as chromogenic substrates . the histochemical staining was performed using the vectastain elite abc kit ( vector laboratories ) . embryos were mounted in canada balsam ( serva ) and photographed with a prog / res/3008 digital camera ( kontron electronic ) on a zeiss axioskop microscope with differential interference contrast optics . gene expression analysis was performed as described , using a custom - designed drosophila oligonucleotide array ( roez003a ; affymetrix ) . the genes represented on the array and considered in this study correspond to 1,513 sequenced drosophila genes encoding open reading frames deposited in swiss - prot / trembl databases as of spring 1998 . for a complete list of these genes each gene is represented on the array by a set of 20 oligonucleotide probes ( 25mers ) matching the gene sequence . to control the specificity of hybridization , the same set of probes , containing a single nucleotide mismatch in a central position , is represented on the array . the difference between the perfect match hybridization signal and the mismatch signal is proportional to the abundance of a given transcript and calculated as its average difference value ( avg diff ) . drosophila genes that were not unambiguously represented by a probe set of 20 probe pairs on the array were excluded from further analysis ( 29 probe sets were not used in this study ) . initial experiments designed to determine the sensitivity and reproducibility of hybridization showed that the use of total rna versus poly(a ) rna as a template for cdna synthesis and subsequent amplification ( synthesis of crna ) gave comparable results , despite the fact that we consistently detected 5s rna and histone genes present on the array with crna derived from total rna . on the basis of these findings , all experiments were carried out using a total rna protocol . total rna was isolated from 200 mg of embryonic tissue , using guanidinium isothiocyanate in combination with acidic phenol ( ph 4.0 ) ( fast rna tube green kit from bio101 ) in a fast prep homogenizer fp120 ( bio 101 ) . after precipitation , the rna was dissolved in depc - treated water ( ambion ) and spectrophotometrically quantified using a genequant rna / dna calculator ( pharmacia biotech ) . cdna was synthesized upon total rna as a template , using the superscript choice system for cdna synthesis ( gibco / brl ) with a t7-(t)24 dna primer . this primer ( 5'-ggccagtgaattgtaatacgactcactatagggaggcgg-(t)24vn-3 ' ) was purified by page . for first - strand cdna synthesis , a typical 40 l reaction contained 25 g rna , 200 pmol t7-(t)24 primer , 500 m of each dntp and 800 units reverse transcriptase ( amv superscript ii ) . second - strand cdna synthesis was carried out at 18c for 2 h in a total volume of 340 l , using 20 units escherichia coli dna ligase , 80 units e. coli dna polymerase i and 4 units rnase h in the presence of 250 m of each dntp . after second - strand cdna synthesis , 0.5 l rnase a ( 100 mg / ml ) ( qiagen ) was added and the samples were incubated at 37c for 30 min . thereafter , 7.5 l proteinase k ( 10 mg / ml ) ( sigma ) was added and the samples were further incubated at 37c for another 30 min . after cdna synthesis was completed , samples were phenol - chloroform extracted , using phase lock gel ( 5 prime-3 prime ) and ethanol precipitated . biotinylated antisense crna was synthesized from the dsdna template , using t7 rna polymerase ( megascript t7 kit : ambion . ) . a 20 l reaction volume contained between 0.3 - 1.5 g cdna , 7.5 mm of both atp and gtp , 5.6 mm of both utp and ctp and 1.8 mm of both biotinylated bio-16-utp and bio-11-ctp ( enzo diagnostics ) and 2 l 10x t7 enzyme mix . the reaction was incubated at 37c for 8 h. thereafter , the unincorporated ntps were removed by running the sample over an rneasy spin column ( qiagen ) . samples were precipitated , taken up in 20 l depc - treated water and spectrophotometrically quantified . thereafter , 40 g of the biotinylated antisense crna was fragmented by heating the sample to 95c for 35 min in a volume of 25 l , containing 40 mm tris - acetate ( ph 8.1 ) , 100 mm potassium acetate , 30 mm magnesium acetate . gene chips were pre - hybridized with 220 l hybridization buffer ( 1x mes ( ph 6.7 ) , 1 m nacl , 0.01% triton , 0.5 g/l acetylated bsa , 0.5 g/l sonicated herring sperm dna ) for 15 min at 45c on a rotisserie ( heidolph ) at 60 rpm . hybridization was done in a final volume of 220 l hybridization buffer , containing 40 g fragmented biotinylated crna . hybridizations were carried out for 16 h at 45c with mixing on a rotisserie at 60 rpm . after hybridization , the arrays were briefly rinsed with 6x sspe - t ( 0.9 m nacl , 0.06 m nah2po4 , 6 mm edta , 0.01% triton ) and washed on a fluidics station ( affymetrix ) . hybridized arrays were stained with 220 l detection solution ( 1x mes buffer , containing 2.5 l streptavidin - r phycoerythrin conjugate ( 1 mg / ml ) ( molecular probes ) ) and 2.0 mg / ml acetylated bsa ( sigma ) at 40c for 15 min and washed again . pixel intensities were measured with a commercial confocal laser scanner ( hewlett packard ) and expression signals were analyzed with commercial software ( genechip 3.1 ; affymetrix ) . detailed data analysis was carried out using race - a ( roche ) , access 97 and excel 97 ( microsoft ) software . for quantification of relative transcript abundance the normalized average difference value ( avg diff ) was used . for each of the three experimental conditions ( wt , hs - wt , hs - lab ) , four replicates were carried out ( for the experimental conditions wt and hs - wt see , including the supplementary data ) . for the difference of the means of the avg diff values over the four replicates between condition 1 ( hs - wt ) and condition 2 ( hs - lab ) , a t - test was performed . moreover , for downregulation , the mean avg diff value of a gene had to be above or equal to 50 in condition 1 ; for upregulation , the mean avg diff value of a gene had to be above or equal to 50 in condition 2 . genes which had a normalized avg diff below 20 obtained automatically an avg diff of 20 ( race - a protocol ) . to obtain a comprehensive analysis of the number and identity of genes differentially regulated by lab , candidates that were already differentially expressed in heat - shocked wild - type embryos compared to non - heat - shocked wild - type controls , were excluded from further analysis ( and data not shown ) . previously , we have used quantiative rt - pcr to confirm that relative expression level changes in the 1.5-fold and above range , as detected on this array , accurately reflect differences in mrna abundance in vivo in drosophila embryos . in consequence , in this report only relative expression level changes in the 1.5-fold and above range are presented . three hundred nanograms of poly(a ) rna , isolated from heat - shocked wild - type embryos and heat - shocked hs - lab embryos ( mrna isolation kit ; roche diagnostics ) , was reverse transcribed with amv - rt and random hexamers ( first - strand cdna synthesis kit for rt - pcr ; roche diagnostics ) . pcr was performed with 100 pg of template dna and gene - specific primers ( designed , using seq web , wisconsin package version 10.0 ; gcg ) on a light cycler ( lightcycler ; roche diagnostics ) . continuous fluorescence observation of amplifying dna was done using sybr green i ( lightcycler - faststart dna master sybr greeni ; roche diagnostics ) . after cycling , a melting curve was produced by slow denaturation of the pcr end - products to check the specificity of amplification . to compare the relative amounts of pcr products , we monitored the amplification profile on a graph , displaying the log of the fluorescence against the number of cycles . relative fold changes for a given gene under both conditions ( heat shock wt versus heat shock hs - lab ) were calculated using the fit point method ( lightcycler operator 's manual , version 3.0 ; roche diagnostics ) . the genes represented on the high - density oligonucleotide array were grouped into 14 functional classes according to the function of the gene product and currently available genetic data . for this , notations in flybase , interactive fly and swiss - prot / trembl databases were used . a comprehensive presentation of all the genes represented on the oligonucleotide array as well as their attribution to functional classes is given as supplementary data to . we thank jan mous , adrian roth , michel tessier , monika seiler and reto brem for essential contributions and helpful advice . we thank clemens broger , martin strahm and martin neeb ( f. hoffman - la roche ) for allowing us to use their race - a chip analysis software and volker schmid and natalie yanze for help with the light cycler . this research was supported by grants from the snsf and the eu - biotech program ( to h.r . ) and by f. hoffmann - la roche . heat - shock - driven ubiquitous overexpression of lab monitored by in situ hybridization and immunocytochemistry . ( a - d ) rna in situ hybridization ; ( e - h ) immunocytochemical staining . expression of lab is shown in heat - shocked wild - type embryos ( a , c , e , g ) and in heat - shocked embryos carrying a hs - lab construct ( b , d , f , h ) . ( a , b , e , f ) overview of stage 10 - 17 embryos . ( c , d ) higher magnification of a single stage 15 embryo and ( g , h ) a single stage 13 embryo ; lateral view , and anterior to the left . embryos were exposed to a heat shock at 36c for 25 min and were allowed to recover for another 25 min before fixation . genes differentially expressed in response to heat - shock - induced overexpression of lab , grouped according to functional classes . bars represent the fold change between differentially expressed genes in heat - shocked wild - type embryos and heat - shocked hs - lab embryos . positive values indicate that the relative expression level of a gene is increased ( upregulated ) following lab overexpression and negative values indicate a decrease ( downregulated ) . absolute average difference ( avg diff ; see materials and methods ) values are given for the lab overexpression condition as follows : white bars represent avg diff < 100 , gray bars represent avg diff ranging from 100 - 1,000 , and black bars represent avg diff > 1,000 . genes differentially expressed in response to lab overexpression genes that are differentially expressed following heat - induced ubiquitous overexpression of lab in stage 10 - 17 hs - lab embryos , grouped according to functional classses . the functional class ' heat - shock proteins ' was excluded from the analysis ( see materials and methods ) . n , number of genes within a functional group present on the chip ; n , number of genes differentially expressed within a functional group following lab overexpression ; n / n 100 , number of differentially expressed genes within a functional class following lab overexpression , given as a percentage of the total number of genes in this class present on the array ; downregulated , total number of genes within each functional class differentially downregulated following lab overexpression ; upregulated , total number of genes within each functional class differentially upregulated following lab overexpression . comparison of fold change between oligonucleotide arrays and rt - pcr rt - pcr was performed on cdna derived from heat - shocked wild - type embryos and heat - shocked hs - lab embryos . fold changes determined by rt - pcr are represented as the mean values of eight independent replicates , derived from two different cdna preparations .

background : homeotic genes are key developmental regulators that are highly conserved throughout evolution . their encoded homeoproteins function as transcription factors to control a wide range of developmental processes . although much is known about homeodomain - dna interactions , only a small number of genes acting downstream of homeoproteins have been identified . here we use a functional genomic approach to identify candidate target genes of the drosophila homeodomain transcription factor labial.results:high-density oligonucleotide arrays with probe sets representing 1,513 identified and sequenced genes were used to analyze differential gene expression following labial overexpression in drosophila embryos . we find significant expression level changes for 96 genes belonging to all functional classes represented on the array . in accordance with our experimental procedure , we expect that these genes are either direct or indirect targets of labial gene action . among these genes , 48 were upregulated and 48 were downregulated following labial overexpression . this corresponds to 6.3% of the genes represented on the array . for a selection of these genes , we show that the data obtained with the oligonucleotide arrays are consistent with data obtained using quantitative rt-pcr.conclusions:our results identify a number of novel candidate downstream target genes for labial , suggesting that this homeoprotein differentially regulates a limited and distinct set of embryonically expressed drosophila genes .

Background Results Discussion Conclusions Materials and methods Fly strains, embryo collections and heat-shock regime Whole-mount High-density oligonucleotide arrays RNA sample preparation and hybridization Data analysis Reverse transcriptase PCR (RT-PCR) Functional classification Acknowledgements Figures and Tables

PMC3125743

in the post - genomic era , with the advent of rapid sequencing techniques , reliable and efficient functional annotation methods are needed . routinely , a translated protein sequence is aligned towards a data base of already annotated sequences and by this it is endowed with different features depending on the level of sequence identity ( si ) . this similarity search is the basis for transfer of annotation by homology . the uniprot knowledgebase ( uniprotkb ; http://www.uniprotkb.org/ ) is presently our major resource of information of protein sequences and of corresponding functions and structures , when available . it provides links also to other resources / data bases , allowing a comprehensive knowledge of experimental and computational characteristics of known / putative proteins and genes . however , only 4.4% of the all protein universe that presently ( uniprotkb release 2011_03 ; 8 march 2011 ) includes some 14 million of sequences has evidence at the protein and at the transcript level . with this scenario , inference of function and structure among related sequences requires the definition of rules to increase the reliability of annotation this is routinely obtained with clustering methods by which sequences are included into sets of similarity . examples of hierarchical clustering include systers ( 1 ) , picasso ( 2 ) and iproclass ( 3 ) . clustr ( 4,5 ) and protonet ( 6,7 ) are the only web servers that comprise the large number of sequences made available by fully sequenced genomes and the entire uniprotkb . both clustr and protonet cluster sequences according to different levels of si , as set by different e - value thresholds , and with different hierarchical algorithms . alternatively , non - hierarchical clustering partitions a sequence data set into disjoint clusters ( 8,9 ) . however , neither hierarchical nor non - hierarchical methods consider explicitly proteins containing multiple domains or proteins that sharing common domains do not necessarily have the same function . proteins with different combinations of shared domains can have different molecular and biological functions , as recently re - discussed ( 10 ) . in order to address these problems , we developed bar ( 11 ) , an annotation procedure that relies on a non - hierarchical clustering method and a large - scale genome comparison where pairs of sequences are selected with very strict criteria of similarity and overlapping of the alignment as described in the next section . we provided statistical validation that bar allows reliable functional and structural annotation in addition to that given by commonly used databases ( 11 ) . here , we introduce bar , an updated and extended version of bar that includes : ( i ) a 5-fold increase in sequences ; ( ii ) go terms from the three main roots ( molecular function , biological process and cellular localization ; http://www.geneontology.org/ ) ; ( iii ) pfam domains ( http://pfam.sanger.ac.uk/ ) ; ( iv ) known ligands and ( v ) for clusters containing pdb structure / s , a cluster hmm model and the corresponding alignment of the target sequence to the optimal template in the cluster for computing its 3d structure . bar is constructed by performing an all - against - all pairwise alignment of all protein sequences ( collected from the entire uniprotkb 05_2010 , with the exclusion of fragments ( 9 399 063 sequences ) , and from the proteome of complete sequenced genomes available on the same date at the national center for biotechnology information ( ncbi ) [ www.ncbi.nlm.nih.gov/genomes/lproks.cgi ( prokaryotes ) ; www.ncbi.nlm.nih.gov/genomes/leuks.cgi ( eukaryotes ) ] and at ensembl ( http://www.ensembl.org/info/data/ftp/index.html ) for a total of 988 complete proteomes ( the list of the species is available at bar+ web site ) . for the sake of comparison , we also used the entire swissprot 03_2011 ( 8 march ) . similarly to bar ( 11 ) , bar is also a non - hierarchical clustering method relying on a comparative large - scale genome analysis . the method relies on a non - hierarchical clustering procedure characterized by a stringent metric that ensures a reliable transfer of features within clusters . in this new version , the method takes advantage of a larger scale pairwise sequence comparison than bar , including 13 495 736 protein sequences . alignment is performed with blast ( 12 ) in a grid environment ( 11 ) . from this we compute for each pair both the si and the coverage ( cov ) defined as the ratio of the length of the intersection of the aligned regions on the two sequences and the overall length of the alignment ( namely the sum of the lengths of the two sequences minus the intersection length ) . each protein is then taken as a node and a graph is built allowing links among nodes only when the following similarity constraints are found among two proteins : their si is 40% and cov is 90% . by this , clusters are simply the connected components of the graph ( 11 ) . seventy percent of the whole data set ( 9 401 223 sequences ) falls into 913 962 clusters . the number of sequence in the clusters ranges from two up to 87 893 in the most populated ( molecular function : abc transporter ) . given our stringent criteria , 87% of the clusters contain sequences whose standard deviation ( sd ) of the protein length is 5 residues . the remaining sequences ( 30% of the total ) originate singletons ( containing just one sequence ) . well annotated sequences are characterized by functional and structural annotations derived from uniprotkb entries ( figure 1 ) . these include go , pfam , pdb and scop ( http://scop.mrc-lmb.cam.ac.uk/scop/ ) ( when available ) . to assess whether go and pfam terms are significant in a cluster , we compute p - values and given the multiplicity of the terms , we applied the bonferroni correction ( 11 ) . we evaluated the cumulative distribution of bonferroni corrected p - values by adopting a bootstrapping procedure . from this we set the threshold p - value at 0.01 in order to discriminate among random and significant ( cluster associated ) features ( 11 ) . validated features ( significant for the cluster ) are those endowed with p 0.01 . according to our procedure when hypothetical and or putative proteins fall into an annotated and validated cluster , they can safely inherit go terms and pfam domain / s even in the case of very low si with the most annotated proteins . these sequences can therefore be labelled as distantly related homologues and inherit function and structure ( when available ) in a validated manner . we previously discussed that this procedure can increase the level of annotation of uniprotkb ( 11 ) . here we increase the level of structural and functional annotations of cluster - included sequences by 54% ( figure 2a ) . they can anyway carry along information ( figure 2b ) , provided that each singleton is endowed with pdb and/or pfam and/or go annotation . our method collects sequences from the protein universe ( uniprotkb ) including also some 988 genomes . by this , all the features [ pdb ( scop classification ) ( red circles ) , go terms ( including molecular function , biological process and cellular localization ) and pfam models ( blue circles ) are also included . an extensive blast alignment is performed of all the 13 495 736 sequences in a grid environment . the sequence similarity network is built by connecting two sequences only if their si is 40% with an overlapping cov 90% . about 913 762 clusters are obtained by splitting of the connected components . by this , any cluster may contain from 2 up to 87 893 sequences ( one cluster containing abc transporters from prokaryotes , eukaryotes and archaea ) . when clusters are endowed with pdb template / s , a cluster - hmm is generated by considering all the sequences that have an identity 40% and a cov 90% with the structure / s ( pink subset ) . the cluster - hmm can be used to align all the other sequences in the cluster to template / s . figure 2.different types of annotations are possible with bar . after clustering and depending on the features ( structure , domains and function ) annotated in the cluster , the percentage of sequences endowed with a given annotation type and inheriting validated annotation ( p < 0.01 ) is indicated . percentage is computed with respect to 9 401 223 comprised in 913 762 clusters . inherited : sequences that inherit annotations by falling into a cluster . without validated annotation : the slice comprises sequences with no annotation and not validated annotations . our method collects sequences from the protein universe ( uniprotkb ) including also some 988 genomes . by this , all the features [ pdb ( scop classification ) ( red circles ) , go terms ( including molecular function , biological process and cellular localization ) and pfam models ( blue circles ) are also included . an extensive blast alignment is performed of all the 13 495 736 sequences in a grid environment . the sequence similarity network is built by connecting two sequences only if their si is 40% with an overlapping cov 90% . about 913 762 clusters are obtained by splitting of the connected components . by this , any cluster may contain from 2 up to 87 893 sequences ( one cluster containing abc transporters from prokaryotes , eukaryotes and archaea ) . when clusters are endowed with pdb template / s , a cluster - hmm is generated by considering all the sequences that have an identity 40% and a cov 90% with the structure / s ( pink subset ) . the cluster - hmm can be used to align all the other sequences in the cluster to template / s . different types of annotations are possible with bar . after clustering and depending on the features ( structure , domains and function ) annotated in the cluster , the percentage of sequences endowed with a given annotation type and inheriting validated annotation ( p < 0.01 ) is indicated . percentage is computed with respect to 9 401 223 comprised in 913 762 clusters . inherited : sequences that inherit annotations by falling into a cluster . without validated annotation : the slice comprises sequences with no annotation and not validated annotations . in bar , when pdb templates are present within a cluster ( with or without their scop classification ) , profile hmms are computed on the basis of sequence to structure alignment and are cluster associated ( cluster - hmm ) ( figure 1 ) . when different templates are present in a cluster the structural alignment among them is computed with mustang ( 13 ) . multiple alignments comprising all the overlapping templates and the sequences similar to them ( with si 40% and cov 90% ) are computed with muscle ( 14 ) and fed to hmmer 2.3 ( 15 ) in order to train the profile - hmm . by this , a library of 10 858 hmms is made available for aligning even distantly related sequences to a given pdb template / s . the server also provides the pairwise query sequence structural target alignment computed with the viterbi decoding implemented in hmmer from the correspondent cluster - hmm and useful for further processing and/or computing the corresponding 3d structure . bar allows 35 possible fine grain types of annotations ( plus no annotation ) ( table 1 ) . the most complete type of annotation is the one with pdb ( with and without scop annotation ) and go terms and pfam domains with p 0.01 ( validated ) ( first row in table 1 ) . interestingly , enough 0.11% of the total sequences in our database are sufficient to annotate in a validated manner and with the most complete annotation another 21.99% sharing common clusters ( 8251 ; 0.90% of the total ) , with an annotation gain factor higher than 200 . summing up ( along the first row of table 1 ) , we can conclude that validated functional annotation is possible within 10% of the clusters . eleven percent of the sequences remains without annotation and are included in 45% of the clusters . about 57% of singletons ( corresponding to 17% of the total set ) table 1.the fine grain types of annotation with barpdb ( % ) scop monoscop multiwithout pdbgo validated pfam validated clusters8251 ( 0.90)3613 ( 0.40)1461 ( 0.16)83 266 ( 9.11 ) sequences2 982 449 ( 22.10)1 408 542 ( 10.44)1 028 565 ( 7.62)2 903 431 ( 21.51 ) inherited2 967 743 ( 21.99)1 404 011 ( 10.40)1 026 154 ( 7.60)1 382 310 ( 10.24 ) pfam clusters8334 ( 0.91)3647 ( 0.40)1463 ( 0.16)85 886 ( 9.40 ) sequences2 984 057 ( 22.11)1 409 647 ( 10.45)1 028 569 ( 7.62)2 922 876 ( 21.66 ) inherited2 969 285 ( 22.00)1 405 095 ( 10.41)1 026 156 ( 7.60)1 398 603 ( 10.36 ) without pfam clusters320 ( 0.04)123 ( 0.01)256251 ( 0.68 ) sequences42 202 ( 0.31)15 415 ( 0.11)7363 ( 0.05)143 533 ( 1.06 ) inherited41 825 ( 0.31)15 303 ( 0.11)7331 ( 0.05)93 568 ( 0.69)go pfam validated clusters8938 ( 0.98)3887 ( 0.43)1504 ( 0.16)133 895 ( 14.65 ) sequences3 042 649 ( 22.55)1 450 437 ( 10.75)1 029 707 ( 7.63)3 311 421 ( 24.54 ) inherited3 026 916 ( 22.43)1 445 521 ( 10.71)1 027 219 ( 7.61)1 617 763 ( 11.99 ) pfam clusters9357 ( 1.02)4033 ( 0.44)1526 ( 0.17)322 937 ( 35.34 ) sequences3 045 465 ( 22.57)1 451 928 ( 10.76)1 029 755 ( 7.63)3 739 076 ( 27.71 ) inherited3 029 337 ( 22.45)1 446 890 ( 10.72)1 027 247 ( 7.61)1 852 223 ( 13.72 ) singletons2608 ( 0.02)1051 515 720 ( 11.23 ) without pfam clusters452 ( 0.05)176 ( 0.02)3045 539 ( 4.98 ) sequences46 311 ( 0.34)17 020 ( 0.13)7400 ( 0.05)330 354 ( 2.45 ) inherited45 803 ( 0.34)16 862 ( 0.12)7362 ( 0.05)226 500 ( 1.68 ) singletons27922129 212 ( 0.96)without go pfam validated clusters679 ( 0.07)345 ( 0.04)1554 314 ( 5.94 ) sequences44 172 ( 0.33)27 775 ( 0.21)654547 459 ( 4.06 ) inherited43 416 ( 0.32)27 410 ( 0.20)633221 585 ( 1.64 ) pfam clusters779 ( 0.09)377 ( 0.04)16122 236 ( 13.38 ) sequences44 582 ( 0.33)27 983 ( 0.21)656695 684 ( 5.15 ) inherited43 735 ( 0.32)27 592 ( 0.20)634301 792 ( 2.24 ) singletons20510702 834 ( 5.21 ) without pfam clusters270 ( 0.03)83 ( 0.01)5412 192 ( 45.11 ) sequences5308 ( 0.04)1771 ( 0.01)1541 494 443 ( 11.07 ) inherited5023 ( 0.04)1689 ( 0.01)149 singletons129101 743 526 ( 12.92)percentage is evaluated with respect to the total number of sequences in the data base ( 13 495 736 sequences ) . bold character : sequences that inherit the annotation typevalues are negligible . validated : p 0.01 ( see text for details , 11 ) . within bar clusters , 35 different types of annotations are possible : ( i ) + go+pfam+pdb [ with or without scop ( monodomain , multidomain ) * ] ; go and pfam are or not validated ( no . of levels = 12 ) . ( ii ) + pfam+pdb ( with or without scop ) * ( no . of levels = 6 ) . ( iii ) + go+pdb ( with or without scop ) * ( number of levels = 6 ) . ( iv ) + pfam+go ( no . of levels = 4 ) . ( v ) + pdb ( with or without scop ) * ( number of levels = 3 ) . ( vi ) + go ( no . of levels = 2 ) . ( vii ) + pfam ( no . of levels = 2 ) . seventy percent of the initial set fall into clusters ( 913 962 ) and 53% in validated clusters . some 6% of the sequences are annotated without validation and the remaining 11% are not annotated ( rightmost bottom cell ) . about 17 and 13% of the sequences are singletons with and without annotations , respectively . the fine grain types of annotation with bar percentage is evaluated with respect to the total number of sequences in the data base ( 13 495 736 sequences ) . bold character : sequences that inherit the annotation type values are negligible . validated : p 0.01 ( see text for details , 11 ) . within bar clusters , 35 different types of annotations are possible : ( i ) + go+pfam+pdb [ with or without scop ( monodomain , multidomain ) * ] ; go and pfam are or not validated ( no . of levels = 12 ) . ( ii ) + pfam+pdb ( with or without scop ) * ( no . of levels = 6 ) . ( iii ) + go+pdb ( with or without scop ) * ( number of levels = 6 ) . ( iv ) + pfam+go ( no . of levels = 4 ) . ( v ) + pdb ( with or without scop ) * ( number of levels = 3 ) . ( vi ) ( vii ) + pfam ( no . of levels = 2 ) . seventy percent of the initial set fall into clusters ( 913 962 ) and 53% in validated clusters . some 6% of the sequences are annotated without validation and the remaining 11% are not annotated ( rightmost bottom cell ) . about 17 and 13% of the sequences are singletons with and without annotations , respectively . when a query sequence is submitted , there are three possible outcomes ( figure 3 ) . the sequence can match a sequence already present in the cluster ( or in a singleton ) . by this , non - annotated proteins can inherit functional and structural annotation from other proteins within the same cluster . validated annotations are inherited when clusters are endowed with validated go and pfam ( p < 0.01 ) . the query sequence may then align with any other sequence in bar with the stringent criteria of our procedure and , therefore , find a cluster from where it can safely inherit all the corresponding structural and functional features . alternatively , when the criteria are not met , all the blast matches are returned . provided that the sequence after running blast has a level of si 40% with a cov 90% to any sequence of bar , it is included into a cluster . in the above example , the cluster is well annotated and the sequence inherits all the possible annotations from the cluster including go terms ( 203 ) , pdb / s , ligands , scop and pfam annotations and the cluster - hmm . furthermore in pir format alignment / alignments of the query sequence to the cluster template / s with cluster hmm is / are also provided . provided that the sequence after running blast has a level of si 40% with a cov 90% to any sequence of bar , it is included into a cluster . in the above example , the cluster is well annotated and the sequence inherits all the possible annotations from the cluster including go terms ( 203 ) , pdb / s , ligands , scop and pfam annotations and the cluster - hmm . furthermore in pir format alignment / alignments of the query sequence to the cluster template / s with cluster hmm is / are also provided . this is based on the notion that indeed the bar annotation system increases its capacity only when we add information . this is achieved when proteins with evidence at the transcript and protein level ( e.g. : pdb new files and/or proteins with go / pfam terms ) are included in the system . for example , by comparing uniprotkb 05_2010 with swissprot 03_2011 , we collected some 2445 sequences carrying information according to our criteria ( evidence at protein / transcript level ) . by aligning this set towards bar clusters , about 8% aligns with singletons and only 0.03% of the total number of bar singletons become new clusters ( with two protein sequences ) . another 7% fall into non - validated clusters without affecting the statistical significance of the cluster - specific annotation . we are currently planning to include other annotation resources in order to extend our annotation process with more protein domains and their interactions . d.p . is the recipient of a miur ( ministero istruzione universit ricerca ) fellowship supporting his ph.d . program ; miur - firb ( fondo per gli investimenti della ricerca di base ) 2003/libi - international laboratory for bioinformatics delivered ( to r.c . funding for open access charge : fondo ordinario per le universit ( ffo ) 2010 delivered ( to r.c . and

we introduce bar - plus ( bar+ ) , a web server for functional and structural annotation of protein sequences . bar+ is based on a large - scale genome cross comparison and a non - hierarchical clustering procedure characterized by a metric that ensures a reliable transfer of features within clusters . in this version , the method takes advantage of a large - scale pairwise sequence comparison of 13 495 736 protein chains also including 988 complete proteomes . available sequence annotation is derived from uniprotkb , go , pfam and pdb . when pdb templates are present within a cluster ( with or without their scop classification ) , profile hidden markov models ( hmms ) are computed on the basis of sequence to structure alignment and are cluster - associated ( cluster - hmm ) . therefrom , a library of 10 858 hmms is made available for aligning even distantly related sequences for structural modelling . the server also provides pairwise query sequence structural target alignments computed from the correspondent cluster - hmm . bar+ in its present version allows three main categories of annotation : pdb [ with or without scop ( * ) ] and go and/or pfam ; pdb ( * ) without go and/or pfam ; go and/or pfam without pdb ( * ) and no annotation . each category can further comprise clusters where go and pfam functional annotations are or are not statistically significant . bar+ is available at http://bar.biocomp.unibo.it/bar2.0 .

INTRODUCTION BAR CLUSTER-HMMs DIFFERENT ANNOTATIONS with BAR SUBMITTING A PROTEIN SEQUENCE TO BAR BAR FUNDING

PMC3901969

increases in weight and adiposity at the population level were first observed in high - income western countries . research has linked these body composition changes to the nutrition and physical activity transitions which are characterized by an increased consumption of refined and processed foods and decreased levels of physical activity and are closely associated with social and economic changes impacting urbanization , food systems , labour demands , and transportation choices [ 25 ] . these transitions seem to be occurring simultaneously and low- and middle - income countries are now progressing through them experiencing similar body composition changes to those that have already occurred in high - income countries [ 68 ] . in fact , in the last decade the prevalence of obesity has tripled in several low- and middle - income countries . as a result , obesity and its related chronic diseases are significant public health issues worldwide [ 10 , 11 ] . in addition to the rise in childhood obesity and inactive lifestyles , secular changes in children 's fitness a strong and independent marker of chronic disease risk [ 12 , 13]have been documented . tomkinson and colleagues calculated that the average annual decline in the aerobic fitness of 619-year - olds from five geographical regions ( africa , middle and east asia , australia , europe , and north america ) was 0.36% between 1958 and 2003 . there is also evidence from developed countries supporting the notion that childhood obesity and fitness levels are negatively correlated . whether or not such associations are consistent in developing countries , and whether changes in body composition and fitness at different stages of the nutrition and physical activity transitions reflect those for obesity , requires further investigation . by comparing the body composition and fitness of children living in countries situated at different stages of the nutrition and physical activity transitions , global correlates of childhood obesity can be better understood . by examining the consistency of these correlates across countries , if correlates are similar from one country to the next , it is likely that comparable factors have contributed to the observed changes and that preventive efforts that work in one country may , if appropriately contextualized , be successful in another . thus , intercountry comparisons can serve to raise awareness , guide the development of preventive initiatives , and further our understanding of this public health concern . the objectives of this study were to ( 1 ) compare body composition , aerobic fitness , and muscular fitness measures in children from three countries that currently sit at different stages of the nutrition and physical activity transitions ( canada - end stages , mexico - mid stages , and kenya - early stages ) and ( 2 ) to examine the intercountry differences in the relationships between body composition and fitness measures . the study population consisted of school - aged children from three countries that currently sit at different stages of the nutrition and physical activity transitions ( canada , mexico , and kenya ) . canada represents a high - income country that currently sits at the final stage of the transitions as shifts in diet and physical activity occurred decades ago and considerable efforts have been underway for the past decade or so to reverse obesity [ 2 , 3 , 6 ] . mexico represents a middle - income country that is at the mid - stages of the transitions as changes in dietary intake and physical activity have occurred , but much later than those observed in high - income countries , and only recently has the issue of obesity begun to be addressed [ 2 , 3 , 6 ] . finally , kenya represents a low - income country that is at the early stages of the transitions as shifts in diet and physical activity are only beginning to emerge [ 2 , 3 , 6 ] . canadacanadian participants consisted of a representative sample of 736 children aged 913 years who participated in the canadian health measures survey ( chms ) [ 1618 ] . the chms is a nationally representative cross - sectional survey with data collected from 15 sites across canada between march 2007 and february 2009 . data collection included a combination of a personal interview ( demographic information ) and a visit to a mobile examination centre for the collection of physical measures , including anthropometry and fitness . canadian participants consisted of a representative sample of 736 children aged 913 years who participated in the canadian health measures survey ( chms ) [ 1618 ] . the chms is a nationally representative cross - sectional survey with data collected from 15 sites across canada between march 2007 and february 2009 . data collection included a combination of a personal interview ( demographic information ) and a visit to a mobile examination centre for the collection of physical measures , including anthropometry and fitness . mexicothe study population consisted of a convenience sample of 193 boys and girls from four public schools located in the urban core of guadalajara , mexico . children in grades 5 and 6 ( 1013 years of age ) from the selected schools were invited to participate . trained personnel directly measured body composition and fitness indicators . the study population consisted of a convenience sample of 193 boys and girls from four public schools located in the urban core of guadalajara , mexico . children in grades 5 and 6 ( 1013 years of age ) from the selected schools were invited to participate . trained personnel directly measured body composition and fitness indicators . kenyaparticipants consisted of a convenience sample of 179 school children aged 913 from four schools in kenya . two of these schools were located in urban areas and two were located in rural areas . data were collected at the four schools by members of our research team in november 2008 . body composition and fitness data participants consisted of a convenience sample of 179 school children aged 913 from four schools in kenya . two of these schools were located in urban areas and two were located in rural areas . data were collected at the four schools by members of our research team in november 2008 . body composition and fitness data ethics approval for data collection was granted for all three study populations by respective institutional review boards . informed consent / assent was also obtained from the child participants and their parents or guardians . with the exception of the aerobic fitness measures in mexico and kenya , all body composition and fitness data were collected in each country using comparable equipment according to the canadian physical activity , fitness , and lifestyle appraisal ( cpafla ) . height ( to the nearest 0.1 cm ) and weight ( to the nearest 0.1 kg ) were measured by trained personnel using calibrated stadiometers and scales , respectively . these measures were used to calculate body mass index ( bmi , kg / m ) . subjects were classified into four categories ( underweight , normal weight , overweight , and obese ) according to the international obesity task force age- and sex - specific bmi cut - points [ 20 , 21 ] . triceps skinfolds were measured in duplicate ( or triplicate if measures varied by > 0.4 mm ) to the closest 0.2 mm using harpenden skinfold calipers ( baty international , uk ) . gulick measuring tapes were used to measure the waist circumference , to the nearest 0.1 cm , according to the world health organization and cpafla protocols ( i.e. , midpoint between last floating rib and top of iliac crest in the mid - axillary line ) . a hand dynamometer ( canada : takei scientific instruments , japan ; mexico / kenya : lb9011 senoh , japan ) was used to measure grip strength in kg . the combined maximum score for each hand was calculated . in the canadian population aerobic fitness was measured using the modified canadian aerobic fitness test ( mcaft ) , during which children had to complete one or more 3-minute stepping stages ( up and down steps with increasing intensity ) at predetermined speeds based on their age and sex . children aged 614 years started at what is stage five for women to a maximum of three stages . participants ' heart rate was recorded after each stage , and the test was completed when it reached 85% of their age - predicted maximal heart rate ( 220 age ) . predicted maximal aerobic power ( vo2max ) was calculated for all participants using the pediatric - specific equation vo2max ( ml / kg / min ) = 3.23 ( oc ) 1.31 ( bmi ) + 1.39 ( age ) 49.21 , where oc is the oxygen cost of stepping . other equations suggested specifically for adults using the mcaft were not used as these have not been validated on children [ 19 , 25 , 26 ] . in the mexican and kenyan populations this test involved continuous running by participants between two lines 20 metres apart in time to recorded beeps on a compact disc . the participants continued running between the two lines , turning when signalled by the recorded beeps . each minute , a sound indicated an increase in speed and the beeps became closer together . if children did not reach the line in time for each beep , the child had to run to the line , turn , and try to catch up with the pace within two more beeps . the test was stopped when the child failed to reach the line ( within 2 metres ) for two consecutive ends . the level at which the child ended the test was recorded , and leger 's equation was then used to calculate peak oxygen consumption ( vo2max ) . this test is currently the most widely used aerobic fitness field test within children and adolescents and has been shown to be a reliable and valid method of estimating vo2max in this age group . all analyses were performed using sas version 9.1 ( sas institute , cary , nc , usa ) . pearson correlations were completed between the three body composition measures within each sex and country subgroup . linear regression models were used to examine the associations between the body composition ( bmi , triceps skinfold , and waist circumference ) , aerobic fitness , and muscular fitness variables . regression diagnostics showed that residuals of the dependent variables ( bmi , triceps skinfold , and waist circumference ) were normally distributed , and thus no transformations were needed . differences in the descriptive and regression analyses across countries were determined by examining whether 95% confidence intervals of the means and regression ( intercepts and coefficients ) overlapped . because of the complex sampling strategy , bootstrapping techniques were used on the canadian data to generate the confidence intervals [ 29 , 30 ] . there were no differences in the mean height of girls in all three countries , but the mean height of kenyan boys was less than that of canadian and mexican children . the mean bmi , waist circumference , and skinfold values of canadian and mexican boys and girls were higher than those of their kenyan counterparts . there were no differences between canadian and mexican children for these three body composition measures with the exception of waist circumference , which was higher in mexican boys . the prevalence of obesity was highest in mexican children while the prevalence of underweight was highest in the kenyan children . however , aerobic fitness ( vo2max ) was different in boys across all three countries with the kenyan 's having the highest values and the canadian 's having the lowest values . in girls , table 2 shows the correlations between the three body composition measures within each sex and country subgroup . correlation coefficients were quite strong ( r value range of 0.620.95 ) , irrespective of sex and country . the correlations in kenyan boys and canadian girls tended to be weaker than in the other sex and country subgroups . bmi tended to be more strongly correlated with waist circumference than triceps skinfold , regardless of sex and country . table 3 shows the results from the age - adjusted linear regression analyses looking at the association between aerobic fitness and the three body composition measures . the table displays the slopes ( beta - coefficient ) of the regression lines , the model fit ( r ) , and the predicted bmi at a vo2max of 40 and 50 ml / kg / min for each sex and country subgroup . the overall patterns of findings indicate the following : ( 1 ) aerobic fitness and body composition measures were negatively associated irrespective of country , sex , and body composition measure examined . ( 2 ) the slopes of the regression lines and predicted bmi at a low aerobic fitness ( e.g. , 40 ml / kg / min ) tended to be greater in mexican children than in canadian and kenyan children . thus as illustrated in figure 1 for bmi , mexican children with low aerobic fitness levels had higher body composition values than did canadian and kenyan children . however , the body composition values of children in all three countries were similar in those with high aerobic fitness ( e.g. , 50 ml / kg / min ) . ( 3 ) the r values for both sexes were higher in canadian children ( range 0.370.53 ) than in mexican children ( range 0.310.37 ) and higher in mexican children than kenyan children ( range 0.110.32 ) . thus , aerobic fitness was more strongly associated with obesity in the most developed country ( canada ) and least strongly associated with obesity in the least developed country ( kenya ) . table 4 and figure 2 show the results from the linear regression analyses looking at the associations between the muscular fitness ( grip strength ) and body composition measures . the overall findings indicate the following : ( 1 ) muscular fitness was not associated with any of the body composition measures in boys and girls from kenya . ( 2 ) muscular fitness was positively associated with bmi and waist circumference , but not skinfold thickness , in boys and girls from canada and mexico . these associations were weak ( r range = 0.090.14 ) in canadian children and mexican boys and were of a modest strength ( r = 0.32 ) in mexican girls . the results provide supporting evidence of intercountry differences in the aerobic fitness and body composition of children from countries at different stages of the nutrition and physical activity transitions . negative relationships between aerobic fitness and obesity were observed in boys and girls from all three countries ; however , these relationships were more pronounced in mexican children than in canadian and kenyan children . differences in aerobic fitness were observed across all three countries wherein kenyan children were the most fit and canadian children were the least fit . although mixed results have been reported in the literature ( possibly resulting from the use of invalid self - reported physical activity questionnaires ) , evidence based on valid questionnaires , objective physical activity measures , and physical activity interventions suggest that aerobic fitness reflects the amount of aerobic physical activity performed in recent weeks and months [ 3234 ] . thus , results from the current study are consistent with each country 's current stage within the physical activity transition . these results are also supported by tomkinson and olds who compared the secular decline in the aerobic fitness of 619-year - old children from 27 countries in recent decades . their results showed that the rate of decline in high - income countries was greater than that of middle- and low - income countries ( 0.49% versus 0.39% per year ) . in the current study irrespective of country , grip strength was not related to triceps skinfold thickness ; however , grip strength was a weak positive correlate of bmi in canadian and mexican children . because weight gain is associated with increases in both lean body mass and fat mass , the positive associations observed were likely driven by a greater lean body mass in the heavier children within canada and mexico . we speculate that the positive effects that the increased lean body mass had on muscular fitness in the heavier canadian and mexican children were not reflected in higher grip strength values than in kenyan children because these effects were negated by decreases in physical activity that affected muscle quality ( e.g. , strength per kg of muscle ) . it is also possible that insufficient variability in the bmis of the kenyan sample resulted in a lack of power to detect meaningful associations . the low prevalence of overweight and obesity in the kenyan children ( 5.6% ) examined in this study was expected given their stage of the nutrition and physical activity transitions and previously published data from that country . in particular , the 2003 kenya global school - based student health survey found that only 5.9% of 1015-year - old boys and girls were overweight or obese [ 8 , 36 ] . although mexico sits at an earlier stage of the nutrition and physical activity transitions than canada , the prevalence of obesity in the mexican children ( 9.2% boys , 8.4% girls ) studied here was slightly higher than in the canadian children ( 8.6% boys , 7.2% girls ) . although this observation is inconsistent with where the two countries currently sit within the nutrition and physical activity transitions , this was not unexpected as these differences are consistent with nationally representative data for the two countries . specifically , the prevalence of obesity in 519-year - old boys and girls in the 2006 mexican national health and nutrition survey was between 16.5% and 23.3% while the prevalence of obesity in 617-year - old boys and girls in the 2004 canadian community health survey was between 7.5% and 11.1% . the higher rates of obesity in mexican children may be due to a variety of factors including differences in dietary and physical activity behaviours , biological differences , and how they interact with their environments . growth stunting ( very low height for age ) could also be a plausible explanation for the higher obesity rates observed within the mexican population . for example , between 1988 and 2006 stunting decreased from 27% to 16% in mexican children under the age of 5 and results from the 2006 mexican national health and nutrition survey found that only 9.9% of children between the ages of 5 and 11 were stunted . furthermore , within the current study no differences were observed between the height of canadian and mexican children suggesting that , in the current study , the higher rates of obesity were not likely due to stunting . although temporality of relationships can not be addressed in this study , the relations between the aerobic fitness and body composition measures suggest that low fitness has a greater impact on the body composition of mexican children than on that of canadian and kenyan children . thus , as mexico continues to progress through the physical activity transition , wherein their physical activity and fitness levels approach those currently observed in canada , we can anticipate that the obesity levels in mexican children will rise at a faster rate than what has occurred in canada in recent decades . conversely , as kenya progresses through the physical activity transition , the increased prevalence of obesity in the population may more closely match what has occurred in canada . nonetheless , our findings suggest that it may be inaccurate to project changes in children 's body composition in developing countries based on previous trends observed in developed countries . thus , reproducing preventive physical activity initiatives that have been successful in high - income countries may have varying levels of success in lower - income countries . for example , our findings suggest that more substantial changes in physical activity and fitness would need to occur within mexican children to have the same body composition benefits observed in predominately non - hispanic white populations such as canada . dietary initiatives may also differ ; however , the differential effects of diet on the body composition of children in different countries requires further investigation . as with all studies , because the kenya and mexico testing sites were at schools that did not have a gymnasium , the aerobic fitness testing was performed outdoors where the high temperature and humidity could not be controlled . altitude also negatively impacts aerobic fitness performance , and therefore the vo2max values obtained around the city of nairobi in the kenyan children were likely underestimated ( though this would only further strengthen our findings ) . in addition , the aerobic fitness of canadian children was assessed using the mcaft test as opposed to the 20 m shuttle run test that was used in mexico and kenya . thus , equations used to estimate vo2max were different for canadian youth resulting in possible comparability issues . furthermore , the mexican and kenyan samples were convenience samples , which limit the generalizability of the findings , particularly as it pertains to how they may have been influenced by urban / rural status . approximately 50% of the kenyan sample was from an urban area , while in the country as a whole only 22% of the population is urbanized . the entire ( 100% ) mexican sample was from an urban area , while in the country as a whole 78% of the population is urbanized . even within a country children may sit at different stages of the nutrition and physical activity transitions depending on where they live . in kenya , for instance , children residing in urban areas are more obese and have lower physical activity and fitness levels than children residing in rural areas [ 7 , 8 ] . while this urban / rural issue may have influenced the descriptive data , they were unlikely to have influenced the relations between the fitness and body composition measures that is , when relationships between body composition and fitness measures were assessed by rural and urban dwelling in the kenyan sample , no significant differences were observed in the intercepts and regression coefficients ( data not shown ) . in conclusion , there appear to be differences in the fitness and body composition measures of children from countries that currently sit at different stages of the nutrition and physical activity transitions . while negative relationships between aerobic fitness and obesity were observed in children from all three countries examined in this study , these relationships were more pronounced in mexican children . this may , in part , explain why the prevalence of obesity was higher in mexican children than in their canadian counterparts even though mexico is at an earlier stage of the nutrition and physical activity transitions .

background . the physical activity transition is contributing to an increase in childhood obesity and a decrease in fitness worldwide . this study compared body composition and fitness measures in children from three countries and examined intercountry differences in the relationship between these variables . methods . participants consisted of 736 canadian , 193 mexican , and 179 kenyan children aged 913 years . body mass index ( bmi ) , waist circumference , triceps skinfolds , aerobic fitness , and muscular fitness were measured . linear regression was used to examine associations between variables . results . the prevalence of obesity was the highest in mexican children ( 9.2% boys , 8.4% girls ) and the lowest in kenyan children ( 0.9% boys , 2.8% girls ) . aerobic fitness ( vo2max in ml / kg / min ) was the highest in kenyan children ( 50.2 boys , 46.7 girls ) and the lowest in canadian children ( 41.3 boys , 38.3 girls ) . aerobic fitness was negatively associated with body composition measures irrespective of country and sex . mexican children with low aerobic fitness had higher body composition measures than canadian and kenyan children . muscular fitness was not associated with the body composition measures in kenyan children but was a weak positive correlate of bmi and waist circumference in canadian and mexican children . conclusion . the current study provides some evidence to support the physical activity transition hypothesis .

1. Introduction 2. Methods 3. Results 4. Discussion

PMC1567985

the inhibition of differentiation and persistence of proliferation in cell transformation is probably not only caused by the mutation of single genes . an additional mechanism of transcriptional control , not only of single genes but of gene programs , is possibly the alteration of the topoisomerases . these enzymes regulate the conformation of dna by twisting and unwinding the double strands . as has been shown previously , only the genes located in relaxed dna areas are transcribed and , therefore , the topoisomerases can be described as a gene regulation device . we present the hypothesis that topoisomerase ii action is not only altered in , but also necessary for , hl-60 granulocytic cell differentiation . thus , alteration of topoisomerases may well be a molecular mechanism of cellular differentiation.imagesfigure 1.figure 2 .

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PMC3697410

leishmaniasis is a global zoonosis from the tropics and subtropics , with humans serving as accidental hosts . due to disease prevalence , one - tenth of the world 's populations ( 700 million people ) globally , there are 12 million cases , and the incidences of visceral ( vl ) and cl infections are approximately 0.5 and 11.5 million new cases each year , respectively [ 1 , 2 ] . in a sample population ( n = 162 ) , parasites were obtained from 85 patients ( 52.5% ) , and were isolated from the blood by cultures of 50 patients ( 30.9% ) . isoenzyme analysis confirmed that the organisms in blood and skin were the same , which underlines the invasive potential of the parasite that escaped the skin . extracellular procyclic promastigotes in the vector mature to metacyclic promastigotes ( motile ) , that evolve to amastigotes ( nonmotile ) once they enter cells in the vertebrate host after the insect bite . the amastigote eventually evolves back to the promastigote form in the vector after a blood meal in infected hosts , closing the cycle . the mature infective metacyclic promastigotes have surface glycosyl inositol phospholipid ( gipl ) and lipophosphoglycan ( lpg ) , virulence factors which inhibit the action of the complement system . once inside the host , metacyclic promastigotes are taken up by macrophages through binding to complement receptors 1 and 3 or c - reactive protein receptor and , after 2472 hours , transform into intracellular amastigotes with no surface gipl or lpg . the amastigotes begin to multiply in the parasitophorous vacuole in the macrophage , suppressing interferon gamma ( ifn ) and the production of nitric oxide ( no ) and superoxide . the immunological response in humans and experimental animals is induced by the amastigote form and not by the promastigote , which enters the host target cell immediately after infection and is not seen by the host immune system . amastigotes inhibit antigen presentation by repressing the expression of class i and class ii mhc , both basally and following stimulation with ifn [ 57 ] . the insoluble antigenic fraction from parasites primarily stimulated cd4 + t cells , while the soluble fraction showed a mixed profile , with cd4 + t cells being the main responsible for th2 cytokines and cd8 + t cells for th1 cytokines . residual parasites remain in the host forever and can be reactivated by aids [ 9 , 10 ] . the challenge is to identify antigens and understand how humoral and cellular immune mechanisms cooperate for immunoprophylaxis , immunotherapy , and clinical remission of lesions [ 11 , 12 ] . the control measures are early case detection and chemotherapy which has been hampered by the toxicity of drugs , severe side effects and by drug resistance in parasites . candidate antigens , including killed promastigotes , live attenuated parasites , crude parasites , pure or recombinant leishmania proteins or dna encoding leishmanial proteins , and immunomodulators from sand fly saliva , have been used ; however , very few candidate vaccines have progressed beyond the experimental stage [ 1 , 13 ] . increased synthesis of heat shock protein ( hsp ) occurs in prokaryotic and eukaryotic cells when they are exposed to stress , to protect themselves from lethality , and represent target antigens of the immune response . interestingly vt vaccine also induced clinical remission of psoriasis [ 15 , 16 ] , psoriatic arthritis [ 17 , 18 ] , and rheumatoid arthritis , a serendipity finding . in this paper we present evidence of the immunoprophylactic and immunotherapeutic effects of insoluble proteins from amastigotes , grown in a liquid culture medium without mammalian cells , and the analysis of humoral and cellular immune responses by elisa and immunoblottings in vt vaccinated and glucantime treated volunteers before and after clinical remission of lesions in primary and secondary infections in human beings . the following leishmania strains were used : l(l)amazonensis ( la : ifla / br/67/ph8 ) , l(l)venezuelensis ( lv : mhom / ve/80/h16 ) , l(v)brasiliensis ( lb : mhom / ve/75/h27 ) , and l(l)chagasi ( lch : mhom / br/74/pp75 ) . first generation polyvalent antigens and second generation monovalent antigens ( la , lv , lb , and lch ) vaccines were prepared as published [ 1520 ] . the final first generation polyvalent immunogen contained 1 mg / ml or 250 g of each leishmania specie antigens in pbs supplemented with rehydragel and 4 g / ml of gentamicin . the concentration of alumina was 0.25 ml per mg ( v / w ) of parasitic protein . cases with ulcers or nodules in patients from el ingenio and la planta from january , 1994 to august , 2000 were considered as potential cl patients detected in house per house visits every 15 days . diagnosis was established by idr > 5 mm with polyvalent leishmanine and elisa with amastigotes antigens . concomitant bacterial infections in ulcers were treated with flucoxaciline ( floxapen ) at 100 mg / kg / day orally every 8 hours for 10 days . patients with idr > 5 mm were selected , a biopsy taken with dermal punch of 6 mm in diameter , from the edge of the ulcer , and aliquots processed for microscopic analysis of amastigotes in macrophages after may - grunwald - giemsa staining , injection of 0.1 ml in mice footpads , and parasite culture in o'daly 's medium . peripheral blood from patients was also taken by venous puncture in the left arm , with vacutainer for elisa and immunoblottings . an effective clinical remission was considered the total involution of ulcers or nodules and the appearance of a retractile scar without any inflammatory signs and no relapse for a follow - up period of 6 months after remission . the study group population ( n = 87 ) was in average 25.1 17.1 years old range 5 to 62 years old 45.5% were females and was distributed in several group samples that received different treatments as explained in tables 1 , 2 , and 4 . immunoprophylaxis ( vt ) was applied with amastigotes proteins to susceptible volunteers by personnel from the ivic immunobiology laboratory , in el ingenio and la planta in guatire miranda state , venezuela , from october to december , 1993 . polyvalent first generation vaccines were injected intramuscularly in the deltoid area in 0.20 ml of pbs containing 200 g of amastigotes proteins monthly , for three times , one month apart , with previous signature of informed consent . for immunotherapy , volunteers with cl from january , 1994 to august , 2000 with lesions > 3 months evolution , received 500 g / dose of vt , weekly , up to 12 doses intramuscularly in the deltoid region . house per house visit was done every 15 days for finding efficacy of treatment ( tables 1 , 2 , and 4 ) . glu treatment was done at the tropical medicine institute , central university of caracas ( ucv ) , venezuela , doses calculated at 10 mg / kg / day and injected during 10 days , with a follow up every 15 days . eligible subjects included males and females 5 to 62 years of age . for immunoprophylaxis subjects were idr and with no previous cl . additional inclusion criteria were use of a medically accepted form of contraception throughout the study and a negative pregnancy test ( females of child - bearing potential ) . for immunotherapy , idr+ volunteers with lesions present for at least 3 months were diagnosed and treated as explained above . any female subject pregnant or lactating all trial participants were evaluated for a history of documented immunodeficiency , hiv status , opportunistic infections , or ongoing uncontrolled infections and were a basis for exclusion . subjects , whose medication involved any vaccines , allergy desensitization products , or use of topical therapy ( except emollients ) for cl within 2 weeks preceding the first administration of the study medication , were excluded from trial participation . subjects with a known hypersensitivity to gentamicin or any other ingredient in the study medication , local anesthesia , or diagnostic agents used for tests pertaining to the trial protocol and a documented history of alcohol abuse were excluded from trial participation . the idr skin test with leishmania antigens provides a dth reaction and is a good marker of the cellular immune response in leishmaniasis . leishmania skin test solutions ( polyvalent leishmanine ) were prepared with equal concentrations of four leishmania species ( la , lv , lb , and lch ) . also , monovalent leishmanine was prepared for each leishmania species . the final concentration of the solution in each case was 40 g / ml in pbs . four g in 0.1 ml was injected intradermally in the volar surface of the right arm . the reaction was measured 48 hours later by the ball - point - pen technique . 50 g of each leishmania specie for coomassie blue staining and 60 g for silver staining were electrophoresed in invitrogen 420% nupage tris - acetate sds - page precast gels under reducing conditions with tris - glycine sds running buffer in a xcell surelock mini - cell ( invitrogen life technologies ) following the manufacturer 's instructions as published [ 16 , 19 , 23 ] . proteins were stained with silverxpress silver or coomassie brilliant blue ( r-250 ) staining kits ( invitrogen ) and analyzed in a biorad image system with quantity one 1d analysis software . antileishmania antibodies to amastigotes in sera from volunteers were performed before and after treatments , as published [ 17 , 19 , 20 , 23 ] . the antigens were derived from amastigotes of the four leishmania species that constitute vt drug substance , tested as polyvalent extracts , and separately not pooled , as found in the vt drug product ( la , lv , lb , and lch ) . the wells of each elisa plate were coated with a solution containing 200 g of antigens per ml . after drying , plates were washed with pbs containing 0.05% tween 20 and postcoated with pbs containing 0.05% tween 20 and 1% bovine serum albumin . the plates were washed , and serum samples ( diluted 1 : 1,000 ) in pbs were added to each well and incubated . another wash was followed by the addition of antihuman igg horseradish peroxidase linked ( fab)2 fragment from sheep and incubation overnight . a final wash was followed by the addition of the substrate abts ( 2 - 2-azino - di - benzothiazoline sulfonate ) and 10 l h2o2 . the assay was read in an automatic elisa reader ( titertek multiskan plus ) at 450 nm . a bovine serum albumin calibrator diluted from 6.25 to 400 ng / ml was added to assigned wells . any replicate wells with an average of 3 standard deviations above the average concentration recorded for the negative controls ( 100 ng / ml ) , was considered a positive reaction . all values represent the average of duplicate assays of three different experiments performed with sera of patients and healthy controls idr. protein concentration was determined by lowry or bca [ 24 , 25 ] . 420% nupage tris - acetate sds - page precast gels under reducing conditions with tris - glycine sds running buffer in a xcell surelock mini - cell ( invitrogen life technologies ) as published [ 17 , 19 , 20 , 23 ] . afterwards transfer to nitrocellulose paper ( schleicher & schull , keene , nh , usa ) was performed in a mini - trans - blot ( biorad ) following manufacturer 's instructions . after reaction with the primary antibody from various patients ' sera and controls , staining was done with a secondary anti - human immunoglobulin horseradish peroxidase linked ( fab)2 fragment from sheep , ( amersham , uk ) at 1 : 1000 dilution for 2 hours at room temperature . finally after a wash with sst ( tris - hcl 0.05 m , 0.15 m nacl , ph 9.5)-tween 20 , a solution with 0.05% diaminobenzidine ( w / v ) , 0.03% h2o2 ( v / v ) , and 0.03% cocl was added , waiting until color was developed , washed with distilled water , and photographs were taken . immunoblottings after staining were aligned side by side from each patient 's sera , digitalized with a scanner scan jet 4c and deskcan ii , 2.3 program ( hewlett packard 19911995 ) and then analyzed with gel - pro 98 , 3.0 media cybernetic 1995 - 1996 . the integrated optical density ( iod ) values corresponding to the area under the curve from different bands was obtained and coefficient of variation calculated before and after clinical remission as follows : od posttreatment / od pretreatment . values > 1.0 revealed increase in od antigens and values < 1.0 decrease in od antigens after treatment . od was proportional to igg primary antibody from sera bound specifically by each leishmania spp . antigens between 13 and 150 kda and revealed by sera from patients and controls with secondary mouse anti - igg ( fab)2 antibody . treatment groups were compared using an analysis of variance ( anova ) model with treatment as a fixed effect . to control for multiple comparisons , if the treatment effect p value was < 0.05 , then pairwise comparisons of each active treatment group versus control were performed using contrasts . the anova assumption of normality and homogeneity of variances was tested , and , where appropriate , a nonparametric approach ( wilcoxon test , tukey 's multiple comparison tests ) was used to compare treatment groups . the study group sample ( n = 87 ) from la planta a hyperendemic region for cl , prevalence 24.8 o / oo , had lesions in uncovered areas from the body remitting with different treatments in six years of followup . similar percentages of patients had scars ( 49.25% ) and ulcers ( 49.25% ) after primary infection , while 1.5% had skin nodules . the ulcer 's area had average 8.62 7.65 cm with a range from 0.44 to 33.18 cm . 170 primary lesions were distributed in the study group as in table 1 ; their evolution had average 2.37 3.32 months , range from 15 days to 9 months . treatments were distributed in 87 patients with primary lesions as follows : 35% had sr of lesions , 42% received glu , and 21% vt vaccine . seven weeks was chosen as golden standard for remission time , selected from patients with natural sr of cl without any treatment . patients treated with glu had 9 weeks and with vt 7 weeks for remission , similar to the golden standard ( table 1 ) . two patients without remission post - glu received 7 1 vt doses and remitted in 7 weeks . patients who received vt had 6 doses for total remission in 7 weeks , without relapses in 6 years of followup ( table 1 ) . patients with secondary infections ( n = 32 ) had mean age 24.69 19.06 years old , range 5 to 62 years old ; 46.87% were females , and scars were found in all patients . time between remission and relapses had average 30.14 27.43 months and range 1111 months . in four patients relapses appeared at the edge of the scars induced by the primary infection . forty scars had total area 7.94 7.38 cm , range 1.930.0 cm , unique scars 65% , and multiple scars 35% , mainly in the right leg . together with scars unique ulcers ( 75% ) predominated over multiple ulcers ( 12.5% ) and unique nodules ( 9.4% ) , over multiple nodules ( 3.1% ) with similar percentages in arms and legs ( table 2 ) . in secondary infections , four patients with new lesions had sr time in 8 weeks , and 7 patients treated with glu had also remission in 8 weeks ( table 2 ) . the four patients with sr had relapses , three had sr again in 4 weeks , one needed 10 glu doses and remitted in 10 weeks . in the group of 7 patients treated with glu in secondary infections , 4 had relapses that remitted without treatment in 6 weeks , and 3 needed 25 new doses of glu remitting in 6 weeks ( table 3 ) . cellular immunity was analyzed by dth response in skin , comparing the idr , between active and sr of lesions . the cutoff point to consider a reaction as positive was diameter > 5 mm . idr was higher in active secondary infections and significantly different to patients with active primary infections ( p < 0.001 ) , while in patients of post - clinical remission values were similar ( p = 0.96 ) . in 127 healthy volunteers without previous cl we found 22.04% idr positivity in the study group ( table 4 ) . the vaccinated volunteers idr exhibited higher idr post - vt ( p < 0.001 ) while idr was similar in idr+ volunteers ( p = 1.0 ) . nonvaccinated controls had higher idr in idr+ volunteers similar to data above ( table 5 ) . the diagnostic value ( specificity + sensitivity ) was 1.76 for patients with active infection and 1.81 post - clinical remissions , which underlines the fact of dth as positive tool for diagnosis of cl with polyvalent amastigotes antigens leishmanine ( la , lv , lb , and lch ) after tlck treatment and np-40 extraction . majority of cl cases ( 59% ) with parasitological diagnosis were in sixty - nine idr nonvaccinated volunteers . in this group in idr+ , nonvaccinated volunteers five cl cases ( 27% ) were found , four remitted spontaneously and only one received glu for clinical remission . nine cl cases ( 14% ) were found in sixty - one idr post - vt vaccinated volunteers , 4 patients had sr and 5 received glu treatment . in ten idr+ post - vt vaccinated volunteers three cl cases were found , one received glu treatment only , and the other two remitted spontaneously . all patients were observed in six years of followup ( table 6 ) . the cutoff point to consider a value as positive was established as the average od in idr( ) controls+3 standard deviations as follows : la = 0.41 , lb = 0.40 , lv = 0.44 , and lch = 0.41 . in primary infections , humoral response was higher with sera from patients with active cl than after sr ( p < 0.001 ) of lesions , and the latter values are all equal or below the cutoff point . the diagnostic value in active infection was la = 1.81 , lv = 1.84 , lb = 1.50 , and lch = 1.84 , evidence that monovalent antigens could be used in elisa as a diagnostic tool for cl ( figure 1 ) . la : l(l)amazonensis ; lv : l(l)venezuelensis ; lb : l(v)brasiliensis ; lch : l(l)chagasi . od values between active pre - sr and pre - vt were not statistically significant ( p > 0.05 ) and were higher than values post - clinical remission of lesions . similar values were obtained in patients with remission in post - sr , post - vt and post - glu treatment ( p > 0.05 ) , which confirmed that treatments with vt or glu were effective and similar to natural sr ( golden standard ) in cl patients . interestingly , od values post - glu in patients that had no remission of lesions were higher than post - sr , post - vt or post - glu with remission of lesions ( p < 0.05 ) , pointing out elisa as a good tool to follow up evolution of cl infection ( figure 2 ) . no significant correlation was found between idr and elisa values in 32 patients with cl . the correlation indexes were for la : 0.0098 ( p = 0.588 ) ; lv : 0.0057 ( p = 0.88 ) ; lb : 0.012 ( p = 0.535 ) ; lch : 0.026 ( p = 0.372 ) . elisa with sera from patients with other tropical diseases showed that in toxocariasis , cisticercosis , and ascaridiasis od values versus controls were not significant ( p > 0.05 ) . on the contrary , sera from chagas disease and/or active cl had higher significant ( p < 0.05 ) values versus controls with all leishmania amastigotes antigens . od values post - clinical remission of cl were lower than active cl , close to the cutoff value , similar to data above ( figure 3 ) . another tool to define important antigens in cl is the analysis of bands ' frequency in immunoblottings of several leishmania species , with sera from active infection and after clinical remission of lesions . antigens 13 to 145 kda revealed by 80100% sera were selected as the most immunogenic proteins . a band was considered present if it had an optical density > 10 units by densitometry analysis . antigenic bands revealed by sera from patients with active cl and post - clinical remission in the same patients were compared within the same group with idr( ) controls ( i.e. , la in active or remission versus la in idr controls , lb in active or remission versus lb in idr controls , and so on with lv and lch ) . common bands present in active , remission , and control idr groups are in red bold numbers . mw bands revealed by sera from active and clinical remission patients , not found by sera from controls , at the respective homologous leishmania specie are in bold blue numbers ( table 5 ) . this is evidence that amastigote antigens sensitized b cells receptors and expanded specific parasites ' clones in active and after clinical remission of lesions not revealed by healthy control sera . total number of antigens decreased postclinical remission in all species as compared to patients with active lesions ( table 5 ) . the shared common antigens by controls and patients explain the location of intracellular amastigotes in the vertebrate host , since promastigotes are immediately recognized by normal immunoglobulin after injection of parasites by the vector in the skin and afterwards phagocytose by apc , neutrophils , and macrophages . specific amastigotes antigens not found by control sera and recognized by sera from active lesions were as follows : 8 in la ; 11 in lb ; 4 in lv ; 6 in lch . in bold green numbers specific antigens found in one leishmania specie only , had the following mw in active lesions : la : 132 ; lb : 115 kda ; lv : none ; and lch : 128 kda ; after clinical remission in the group mw were : la : 100 ; lb : 108 ; lv : 80 ; and lch : 86 kda , respectively . in bold black numbers antigens mw 45 kda were found in controls in la , lb , lv , and lch by 100% sera , also mw 96 kda in lb by 100% control sera . both antigens were not recognized by any patient 's sera ( table 7 ) , only by healthy controls . in normal back numbers , mw 86 kda in la and lv , 90 kda in la and 128 , and 132 kda in lb are in 80100% controls sera but not in sera from cl patients in the respective leishmania group . bands frequency in the sr natural process revealed by sera from four volunteers showed fourteen antigens ( 56% ) mw 142 , 132 , 128 , 120 , 115 , 104 , 100 , 98 , 90 , 86 , 80 53 , 45 , and 27 kda decreasing after remission of lesions . only one antigen ( 4% ) mw 33 kda increased in band frequency ; the rest ten antigens ( 40% ) mw 136 , 108 , 74 , 65 , 60 , 57 , 50 , 40 , 35 , and 22 kda frequency remained unchanged . band frequency before and after glu revealed by sera from three volunteers , showed twelve antigens ( 41.4% ) 145 , 132 , 128 , 104 , 100 , 70 , 53 , 33 , 31 , 24 , 17 , and 15 kda decreasing while four antigens ( 13.8% ) 83 , 80 , 57 , and 29 kda increased after remission of lesions , the rest thirteen antigens ( 44.8% ) 98 , 86 , 78 , 74 , 65 , 60 , 50 , 43 , 40 , 35 , 27 , 22 , and 20 kda remained unchanged . band frequency before and after vt revealed by sera from 3 volunteers showed twelve antigens ( 37.5% ) 142 , 120 , 96 , 80 , 57 , 35 , 29 , 22 , 20 , 17 , 15 , and 13 kda decreasing while two antigens ( 6.25% ) 90 and 60 kda increased after remission of lesions ; the rest of eighteen antigens ( 56.25% ) 145 , 136 , 132 , 128 , 122 , 115 , 104 , 100 , 98 , 86 , 74 , 65 , 50 , 45 , 40 , 33 , 27 , and 24 remained unchanged . decrease in bands ' frequency predominated in low mw antigens from 57 to 13 kda in post - glu and post - vt treatment in comparison to natural sr . molecular weights bands 80 to 145 mw also decreased , eleven antigens post - sr , followed by six post - glu and four post - vt ( table 8) . coefficient of variation was calculated as ( 1)area curve od post - treatmentarea curve od pre - treatment . integrated optical density ( iod ) area values > 1.0 correspond to increase in antigen density , while values < 1.0 corresponds to a decrease in density , respectively . optical density was proportional to igg primary antibody from sera pre- and posttreatment , bound by each leishmania specie amastigotes antigens between 13 and 145 kda , and revealed with secondary mouse antihuman igg ( fab)2 antibody . the iod from amastigote antigens revealed by sera from sr decreased significantly in quantitative immunoblottings in primary ( 100% antigens ) and secondary ( 94% antigens ) infections , after clinical remission of lesions , due to low antibody concentration once the active infection was over and parasites under control . sera from sr had only one antigen mw 70 kda with iod increasing postclinical remission in secondary infections . sera post - glu in secondary infections had increased iod area in six antigens ( 26.1% ) mw : 78 , 65 , 57 , 50 , 40 , and 29 , and the rest seventeen antigens ( 73.9% ) decreased in area density ( figure 4 ) . after primary infection in vt vaccinated volunteers , ( 1 ) sera from previous idr volunteers had decreased in iod area in twenty - eight antigens ( 90.3% ) , and three antigens ( 9.7% ) mw 145 , 122 , and 22 kda increased only . ( 2 ) sera from previous idr+ volunteers with subclinical infections had seventeen antigens ( 54.8% ) which decreased iod values while in the rest fourteen antigens ( 45.2% ) , valuesin iod area increased . ( 3 ) sera idr+ with previous healed cl had decrease in iod in sixteen antigens ( 51.6% ) while the rest fifteen antigens ( 48.4% ) had increase in iod ( figure 5 ) . hamsters immunized with tlck treated lb amastigotes from culture , infected with hamsters lb amastigotes presented ( 1 ) a gradual increase in t and b cell responsiveness to mitogens by lymph node lymphocytes , with an increased response to concanavalin a ( cona ) ; ( 2 ) no changes in response to dextran sulphate and pokeweed mitogen in splenocytes ; ( 3 ) absence of parasites in lymph nodes after 6-week postinfection and a nodule 4 times smaller than that of infected control animals , which was undetectable 70 days after infection . hamsters preimmunized with tlck treated ld amastigotes from culture did not show suppression of the blastogenic response to mitogens of spleen and lymph node cells after infection with ld amastigotes from hamster 's spleen and survived for more than one year , whereas infected , unimmunized animals died five months after infection . animals preimmunized with culture parasites ( lb or ld ) treated with phenylmethyl sulphonyl fluoride ( pmsf ) and infected with lb or ld amastigotes did not show any protective effect . the idr with polyvalent vt had a diagnostic value > 1.0 for cl surveillance useful to know the prevalence and incidence in endemic and hyperendemic regions . positive idr in residents living in endemic areas without previous cl in skin are defined as subclinic infections . in corguinho , do sul , brazil , an endemic region for lb , 15.7% had positive idr to leishmanine with no previous infections ; also in endemic regions for l(l)peruviana in lima , ancash , and piura in peru , 17.0% idr+ cases were recorded and in northeast ecuador with high prevalence of l(l)guyanensis and l(l)panamensis 17.2% positivity was observed ; all values are in agreement with data in venezuela : 22.0% idr+ volunteers to polyvalent amastigote leishmanine in healthy subjects , with no previous cl infection . the association of protective immunity with idr response and cellular immunity explains the higher incidence of cl in idr ( 59% ) as compared with idr+ ( 27% ) volunteers in the study group . proteases play essential roles in the pathogenesis of leishmaniasis , helping parasites to invade and multiply in mammalian host cells . however , few studies have addressed serine proteases in leishmania infection and their role in host pathogenesis . the level of internalization of parasites into host macrophages after treatment amastigotes with the anti-115 kda antibody was significantly reduced , suggesting that this serine protease probably plays a role in the infection process . the basis of vt vaccine is inhibition of serine proteases with tlck , which induced immune prophylaxis and immunotherapy in cl patients . prohibitin is concentrated at the surface of the flagellar and the aflagellar pole , through which host - parasite interactions occur . vt vaccine does not have prohibitin since amastigotes were extracted with np-40 and supernatant discarded ; thus no antibodies or cellular immunity to surface proteins could be found [ 1720 ] . specific antibodies decreased post - clinical remission of lesions , confirmed by elisa , bands ' frequency , and iod bands ' area in immunoblottings , pointing out to the fundamental role of cellular immunity in controlling infection . three months was the time waited in cl patients to start vt or glu treatments to avoid the self - healing leishmaniasis effect . size of skin ulcers in relapses was lower than in primary lesions ; thus , a vaccine was possible to control cl . vt vaccine had remission time evidence vt amastigotes vaccine was useful for cl treatment . furthermore vt vaccinated subjects had lower cl cases in idr and idr+ volunteers than idr nonvaccinated subjects . the persistence of these specific antibodies and an acute increase in their levels might be a sentinel of a vl relapse . elisa has been used for diagnosis of cl using soluble ( sf ) and membrane enriched ( mf ) antigen fractions obtained from lb . sensitivity was 89.5% for each fraction , and specificity was 89.5% for sf and 93.4% for mf . similar results were obtained with vt vaccine insoluble amastigote antigens from four species lb , ld , lv , and lch . elisa with monovalent amastigotes evidenced that antibodies were induced on b cells in primary active infection and decreased significantly post - clinical remission , once parasites were under control . this underlines that cellular immunity played a key role in control of parasites in secondary infection as evidenced by a high positive dth in active and postclinical remission of lesions . elisa in active lesions , pre - sr and pre - vt was similar in primary infection evidencing that b cell clones were activated and reacting to all monovalent amastigotes . antibodies decreased significantly post - sr , post - vt , and post - glu treatment , since vt was stimulating mainly cellular immunity . interestingly antibodies did not decrease in one group of patients without clinical remission post - glu treatment . the od values in this group , compared with values that had clinical remission , were significant ( p < 0.05 ) , a very good internal control . the reason why these patients could not switch on cellular immunity and decrease humoral immunity is under investigation . microorganisms replicate in macrophages , facilitated by surface igg antibody increasing intracellular infection through fc-receptor by phagocytosis . the ligation of monocyte or macrophage fc receptors by igg immune complexes , suppresses innate immunity , increases production of il-10 , and bias t - helper-1 ( th1 ) responses to th2 responses , leading to increased infectious output by infected cells [ 35 , 36 ] . normal igg antibodies recognized many parasite antigens , isolating them from the immune system , directing promastigotes to the intracellular site . cellular immunity in chronic infection was probably mediated by a cd8 + t cells and th1 response once intracellular amastigotes were under control and lesions disappeared . parasites remain in the host forever , in proliferative equilibrium inside cells , without cells ' lysis , controlled by a permanent cellular immunological response , which once it is stopped , parasites can be reactivated and disseminated as in aids [ 9 , 10 ] . bands frequency analysis revealed decreased numbers of bands in 56% antigens recognized by sera after sr , 41% post - glu , 37% post - vt , respectively , explaining od decreasing in elisa , in sr and after glu or vt treatments . the highest decrease was in low molecular weight antigens post - glu and post - vt treatment . decrease in bands ' frequency and serum antibodies may be explained by inhibition of antigen presentation by repression of class i and class ii mhc on infected host cells [ 5 , 6 ] , by interfering with the loading of antigens onto the mhc class ii molecule or sequestering the mhc ii molecules and antigens within the phagolysosome . the iod area in amastigote antigens revealed by sera from sr volunteers and post - glu treatment decreased significantly , and only one antigen 70 kda increased after sr in secondary infections . sera post - glu had increased iod area in low mw antigens 78 , 65 , 57 , 50 , 40 , and 29 kda ( 26% ) after clinical remission of lesions . this was probably due to parasites ' death and liberation of low mw antigens to the host , inducing b cell sensitization , antibody production , and immune protection , similar to findings in immunized and infected balb / c mice . after vt vaccination , iod area in majority of proteins decreased in idr patients , while low and high mw antigens increased in idr+ subclinical infections , and in idr+cl+ , vt vaccinated volunteers with previous healed cl suggesting vt vaccination also killed parasites inducing nk cells liberating antigens in subclinical and clinical infections , similar to glu treatment . to recognize amastigote antigens , no previous lymphocyte sensitization is required . leishmania promastigotes sonicates induced in vitro proliferation and ifn production in peripheral blood mononuclear cells ( pbmc ) from individuals that never had contact with leishmania parasites . the proliferating t cell population was cd2 + in a frequency < 1 : 10,000 a response that could be abolished after depletion of cd45ro+ memory cells from the pbmc . thus , normal cd2 + t cells and normal immunoglobulin from healthy volunteers that never experienced leishmania infection reacted with leishmania antigens in the immunoblotting assay . interestingly psoriatic patients , dth negatives to amastigote antigens were also recognized in the blastogenic assay leishmania antigens and were distributed in two groups , low and high responders to amastigote antigens before treatment . furthermore , a high dth response with lb and lch protein chromatography fractions in vivo after treatment with vt polyvalent vaccine was found in human volunteers , confirming the absence of immunosuppression . the lymphocyte stimulation indexes obtained by protein fractions were 5070% lower than the values induced by intact living leishmania amastigotes . this suggests in vitro stimulation appears to be selectively focused on a particular subset of lymphocytes which may be regulatory cd8 + t cells [ 1720 , 41 ] . surprisingly , vt amastigotes antigens induced clinical remission of all forms of psoriasis , psoriatic arthritis ( psa ) , and rheumatoid arthritis in human volunteers and collagen induced arthritis in mice a serendipity finding . the effect of vt was analyzed in patients with psa after treatment , finding that c - reactive protein ( crp ) , complement 5a , tnf , and 1l-1 decreased in human volunteers . interestingly , proliferation of cutaneous t cell lymphoma in vitro was also inhibited by vt monovalent lch amastigotes antigens in a dose / response relationship , which open new roads for the application of this vt treatment to wide spectra of diseases . due to genetic diversity in target populations , including both dogs and humans , a multiple - antigen vaccine will likely be essential . however , the cost of a vaccine to be used in developing countries must be considered . the polyprotein ( ksac ) was found to be immunogenic and capable of inducing protection against l(l)infantum , responsible for human and canine vl , and against l(l)major , responsible for cl . it is a cost - effective vaccine capable of protecting both humans and dogs against multiple leishmania species . the vt polyvalent vaccine for cl , ml , and vl has a very low cost of production , is feasible to be applied to developing countries worldwide . hsp long - term confrontation of the immune system similar in the host and microbes invaders may convert the immune response against these host antigens and promote and/or decrease autoimmune diseases including psoriasis [ 4345 ] . there is evidence that recognition of self - hsp60 can have beneficial effects in arthritis and may offer new strategies for improved control measures in the inflammatory processes by administration of peptides cross - reactive to self - determinants . hsp60 , hsp70 , and gp96 function as host derived ligands for toll - like receptors ( tlr2 ) and play a role in the pathogenesis of ra and psoriasis [ 44 , 45 ] . leishmania antigens are produced after a heat shock in promastigotes that become amastigotes in a liquid culture medium . the mw of chromatography fractions from lb that induced remission of psoriasis was similar to the range of most hsp host ligands ( 5070 kda ) and could be inhibiting the symptoms of psoriasis , psa , and cia [ 19 , 20 ] by competing with peptides in the respective receptors . similar mechanisms could be acting in immunoprophylaxis and immunotherapy of cl as shown in this paper . amastigotes from four leishmania species , cultured in a free cells culture medium without cells treated with tlck ( vt ) induced clinical remission of cl after six 500 g / dose , weekly , up to 12 doses intramuscularly in the deltoid region in 7 weeks and protected volunteers from cl in six years of followup . humoral immunity decreased after clinical remission of lesions , measured by antibodies in elisa , frequency of antigenic bands , and density area densitometry in immunoblottings . vt vaccine induced mainly cellular immunity , measured by dth intradermic reaction , postremission of lesions as compared to active infection . vt vaccine induced clinical remission of all forms of psoriasis , psa , and rheumatoid arthritis in human volunteers and collagen induced arthritis in mice , a serendipity finding , and inhibited proliferation of cutaneous t cell lymphoma in vitro which open new roads for the application of this vaccine to wide spectra of diseases .

amastigotes from l. ( l.)amazonensis ( la ) , l. ( l.)venezuelensis ( lv ) , l. ( v.)brasiliensis ( lb ) , and l. ( l.)chagasi ( lch ) were cultured in a free cells liquid culture medium . patients ( n = 87 ) from a cutaneous leishmaniasis ( cl ) hyperendemic region receiving different treatments were followed up from january 1994 to august 2000 . time for remission of lesions were spontaneous remission ( sr ) 7 weeks ; glucantime ( glu ) chemotherapy 9 weeks ; immunotherapy with la , lv , lb , and lch amastigotes tosyl - lysil chloromethyl - ketone ( tlck ) treated and nonidet p-40(np-40 ) extracted ( vt ) 7 weeks . delayed type hypersensitivity ( dth ) response with leishmanine intradermic reaction ( idr ) was higher in cl patients than healthy controls ( p < 0.05 ) and increased in active secondary versus primary infection ( p < 0.001 ) with diagnostic value 1.74 for active infection and 1.81 postclinical remission . antibodies to amastigotes characterized by enzyme linked immunosorbent assay ( elisa ) decreased in sera postclinical remission versus active infections ( p < 0.001 ) , with a diagnostic value from 1.50 to 1.84 . immunoblottings antigenic bands frequency as well as integral optical density ( iod ) area densitometry decreased with sera from sr , after glu or vt treatments in cl volunteers . intracellular parasitism is due to normal antibodies recognizing parasite antigens after inoculation by vector . vt vaccine induced mainly cellular immunity , for remission of lesions and protection from cl infection .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

PMC3775433

glutamate reuptake represents the principal mechanism for inactivation of synaptically released glutamate [ 1 , 2 ] . in the rodent hippocampus , it is mainly accomplished by astrocytic glutamate transporters ( eaats : excitatory amino acid transporters ) , namely , glast ( glutamate / aspartate transporter ) and glt-1 ( glutamate - transporter-1 ; rodent analogues of eaat1 and eaat2 , resp . ; [ 37 ] ) . glutamate uptake is energized by the concomitant inward transport of three sodium ions and a proton , while one potassium ion is transported outward . consequently , its activation is accompanied by an increase in the intracellular sodium concentration of astrocytes [ 8 , 9 ] . under pathological conditions , astrocytes undergo a complex reaction referred to as reactive astrogliosis , which is seen in diverse preparations and conditions ranging from primary cell culture to the intact brain [ 10 , 11 ] . the hallmarks of reactive gliosis are a massive upregulation of the expression of the intermediate filament glial fibrillary acidic protein ( gfap ) and a cellular hypertrophy [ 12 , 13 ] . reactive astrocytes display several features of immature astrocytes ( e.g. , [ 14 , 15 ] ) and can partially reenter the cell cycle [ 16 , 17 ] . following traumatic brain injury , a dense glial scar forms at the lesion site due to strong astrocytic reorganization and proliferation [ 10 , 11 , 18 ] . at some distance to the lesion and scar , astrocytes usually do not divide , and their morphological reorganization and hypertrophy is less severe . the establishment of a glial scar around injured tissue is regarded as hindrance for the growth and regeneration of axons [ 19 , 20 ] . notwithstanding , reactive astrocytes might also exert a protective role and support regeneration [ 11 , 13 , 21 ] . there is evidence that reactive gliosis is also accompanied by an alteration in the expression level of glial glutamate transporters . most studies performed in the rodent and human brain reported an overall downregulation of protein levels of both glast and glt-1 and/or a reduction in functional glutamate uptake in response to brain injury and astrogliosis , respectively ( e.g. , [ 2227 ] ) . the reduction in the expression of glial glutamate transporters has been suggested to contribute to the elevation of extracellular glutamate concentrations and to glutamate - mediated excitotoxicity which is observed under many pathological conditions . injury - induced changes in the expression of the glutamate transporter subtypes might , however , also depend on the degree of astrogliosis . this might be especially relevant and visible upon a mechanical lesion , where reactivity of astrocytes , as judged for example based on their gfap expression and morphology , ranges from severe astrogliosis in the scar region to only moderate - mild astrogliosis distant from the lesion [ 10 , 11 ] . to study this question , we analyzed expression of glast and glt-1 following a mechanical lesion in organotypic tissue slice cultures of the mouse hippocampus . furthermore , we employed ratiometric sodium imaging as functional assay for glutamate uptake in astrocytes . our results show that mechanical tissue injury generates subsets of reactive astrocytes depending on the distance from the lesion site , which differ in both morphological features and their ability to take up sr101 . furthermore , these subsets show discrete changes in glutamate transporter expression and glutamate uptake capacity , indicating that glutamate clearance might be largely functional in the periphery of the lesion , but strongly hampered in the scar region . this study was carried out in strict accordance with the institutional guidelines of the heinrich heine university duesseldorf , germany , as well as the european community council directive ( 86/609/eec ) . all experiments were communicated to and approved by the animal welfare office at the animal care and use facility of the heinrich heine university duesseldorf , germany ( institutional act number : o52/05 ) . in accordance with the german animal welfare act ( tierschutzgesetz , articles 4 and 7 ) , no formal additional approval for the postmortem removal of brain tissue was necessary . for generation of acute slices , mice were quickly decapitated ( following the recommendation of the european commission published in : euthanasia of experimental animals , luxembourg : office for official publications of the european communities , 1997 ; isbn 92827 - 9694 - 9 ) . acute tissue slices of the hippocampus were prepared from balb / c mice ( mus musculus ) of both genders at postnatal days 7 to 8 ( p7 - 8 ) using standard procedures . for some experiments , transgenic animals ( fvb / n - tg[gfapgfp]14mes / j ) expressing green fluorescence protein ( gfp ) under the gfap promoter were used ( obtained from jackson laboratory ; harbor , usa ) . after decapitation of the animals , brains were quickly excised and hemisected in ice - cold artificial cerebrospinal fluid ( acsf ) composed of ( in mm ) 125 nacl , 2.5 kcl , 2 cacl2 , 1 mgcl2 , 1.25 nah2po4 , 26 nahco3 , and 20 glucose , bubbled with 95% o2 and 5% co2 , and adjusted to a ph of 7.4 . hemisections were trimmed , and transverse slices ( 200 m ) comprising the entorhinal cortex , hippocampus , fimbria , and thalamus were prepared using a vibratome ( microm hm650v , thermo fischer scientific , walldorf , germany ) . organotypic hippocampal slice cultures were prepared and cultured according to the protocol introduced by stoppini et al . with minor modifications . to this end , acute slices were transferred to a millicell culture insert ( picm org 50 , hydrophilized ptfe , pore size 0.4 m ; merck millipore , darmstadt , germany ) and maintained at the interface of a serum - based culture medium free of antibiotics in a humidified incubator atmosphere of 5% co2 at 37c . the culture medium was composed of 30% sterile filtered normal horse serum ( nhs ; gibco / life technologies , darmstadt , germany ) , 30% dulbecco 's modified eagle medium ( dmem ; gibco / life technologies , darmstadt , germany ) , and 40% hank 's balanced salt solution ( hbss ; gibco / life technologies , darmstadt , germany ) supplemented with 38 mm glucose ( ph adjusted to 7.3 - 7.4 ) . the medium was changed three times a week , and the insert surface was washed with medium once a week . after at least 12 days in culture , a mechanical lesion was performed using a sterile scalpel blade . the lesion was positioned in the ca1 area perpendicular to the stratum pyramidale , included the strata oriens , pyramidale , radiatum , and lacunosum moleculare , and spanned the entire depth of the slice ( cf . figure 2 ) . after lesioning , slice cultures were maintained for another 6 - 7 days ( designated as 6 - 7 days postlesion ) . unlesioned control slices were cultured in parallel for a corresponding number of days . for visualization of cell death , acsf containing 0.5 g / ml propidium iodide ( pi ) was applied to the slice surface and incubated for 3 hours at 37c and 5% co2 , followed by a wash with acsf . documentation was either accomplished with an epifluorescence microscope ( nikon eclipse 90i ; nikon instruments , dsseldorf , germany ) or at an olympus fluoview300 laser scanning microscope ( olympus , hamburg , germany ) . astrocyte soma size in organotypic control and lesioned slices was calculated from images of gfp - fluorescence derived from gfp / gfap mice which were obtained at a confocal microscope ( olympus fluoview300 ; olympus , hamburg , germany ; see also below ) . to this end , a semiquantitative approach was used , in which somata of single cells were manually encircled ( cf . figure 4(b ) ) , and the resulting area was calculated using imagej software ( nih , bethesda , usa ) . all chemicals were purchased from sigma - aldrich chemical ( munich , germany ) unless stated otherwise . for imaging experiments , organotypic slices were excised from the millicell inserts and incubated in acsf containing 2.5 m sr101 for 30 min at 35c to stain astrocytes ( see above ) . sr101 is a highly specific and widely used tool for the identification of mature astrocytes in many brain regions including the hippocampus [ 29 , 30 ] . it has recently been shown to be taken up into mature hippocampal astrocytes via an active transport mechanism involving organic anion transport polypeptides . wide - field fluorescence imaging was performed using a variable scan digital imaging system ( till photonics , martinsried , germany ) attached to an upright microscope ( bx51wi , olympus europe , hamburg , germany ) and a ccd camera ( till imago vga , till photonics , martinsried , germany ) . sr101 was excited at 575 nm , and emission was collected above 590 nm . excitation wavelength for detection of gfp was 488 nm , and emission was collected above 510 nm . for intracellular sodium imaging , slices were additionally incubated with the membrane permeable form of the sodium - sensitive fluorescent dye sbfi ( sbfi - am ; sodium - binding benzofuran isophthalate - acetoxymethyl ester ; molecular probes / life technologies , darmstadt , germany ) as described earlier [ 3234 ] . ratiometric sodium imaging was performed by alternate excitation of sbfi at 340 nm ( weakly sodium - sensitive wavelength ) and at 380 nm ( sodium - sensitive wavelength ) at 4 hz . emission ( > 440 nm ) was collected in defined regions of interest ( roi ) representing cell bodies . after background correction , the fluorescence ratio ( f340/f380 ) was calculated for the individual rois and analyzed offline using originpro 8 g software ( originlab corporation , northampton , ma , usa ) . changes in sbfi fluorescence ratio were expressed as changes in sodium concentration based on in situ calibrations as reported before [ 32 , 33 , 35 ] . to equilibrate extra- and intracellular na - concentrations , sbfi - loaded slices were perfused with saline containing ionophores ( 3 m gramicidin d , 100 m monensin ) and the na / k - atpase blocker ouabain ( 100 m ) , as well as different concentrations of na . the glutamate transporter agonist d - aspartate was applied by a pressure application device ( pdes-02d , npi electronic gmbh , tamm , germany ) coupled to standard micropipettes ( hilgenberg , waldkappel , germany ) placed 20100 m from cell bodies of selected cells . the following antisera were utilized , which represent well - established , commercially available standard markers : guinea - pig glt-1 antiserum directed against the c - terminus of rat glt-1 ( chemicon international / millipore corp . , carrigtwohill , ireland ; e.g. , ) and guinea - pig glast antiserum directed against the c - terminus of rat glast ( chemicon international / millipore corp . , carrigtwohill , ireland ; e.g. , ) . validation and a detailed characterization of both antibodies were provided before [ 3840 ] . for identification of astrocytes , polyclonal rabbit antibodies against gfap ( gfap - pab ; dako cytomation , denmark ; [ 40 , 41 ] ) and s100 ( abcam , united kingdom ; ) were employed . when double staining with the latter antibody was performed , a monoclonal mouse antibody detecting gfap was used ( gfap - mab ; sigma aldrich , germany ; [ 40 , 43 ] ) . standard fluorochrome - conjugated antibodies ( alexafluor , invitrogen / life technologies , darmstadt , germany ) were employed as secondary antibodies for immunohistochemistry . prior to immunohistochemical processing , organotypic slices were immersion fixed for 30 min at rt in 4% paraformaldehyde ( pfa ) in phosphate - buffered saline ( pbs ) following three washes every 30 min with pbs . cell membranes were permeabilized , and unspecific binding sites were blocked in pbs containing 0.25% triton - x100 ( tx ) and 2% normal goat serum ( ngs ; gibco / life technologies , darmstadt , germany ) for 90 min at 4c followed by incubation with the primary antibody gfap - pab ( 1 : 1000 ; dako cytomation , glostrup , denmark ) , diluted in the same solution over night at 4c . in case of s100/gfap double stainings , slices were incubated with a mixture of the primary antibodies gfap - mab ( 1 : 1000 , dako cytomation , glostrup , denmark ) and rabbit - s100 ( 1 : 100 , abcam , united kingdom ) . after five washes in pbs containing 0.25% tx and 2% ngs , slices were incubated either with guinea pig - anti - glast or with guinea pig - anti - glt-1 ( both 1 : 1000 , diluted in 0.25% tx/2% ngs / pbs ) for 4 hrs at rt . antirabbit - alexafluor594 and antiguinea pig - alexafluor488 or antimouse - alexafluor488 ( 1 : 100 in blocking solution ) were used for visualization of antibody binding and incubated for 2 hrs at rt . the slices were subjected to dapi staining ( 4,6-diamidino-2-phenylindole ; 0.5 m ; invitrogen ) , washed three times , and mounted on glass slides with mowiol / dabco ( calbiochem , fluka , distributed by sigma - aldrich chemical , munich , germany ) . negative controls were run in parallel to each staining by either omitting all or just one of the primary antibodies . control stainings in which one of the primary antibodies was omitted showed the identical labeling pattern for the remaining antibody as in the double stainings . omitting documentation of immunofluorescence was either performed with an epifluorescence microscope ( nikon eclipse 90i ; nikon instruments , dsseldorf , germany ) or a confocal laser scanning microscope ( olympus fluoview300 ; olympus , hamburg , germany ) . the epifluorescence microscope was equipped with a standard dapi ( ex 340380 ; dm 400 ; ba 435485 ) , fitc ( ex 465495 ; dm 505 ; ba 515555 ) , and tritc ( ex 540/25 ; dm 365 ; ba 605/55 ) filter set . illumination was provided by an intensilight fiber lamp ( c - ghfi ; nikon instruments , dsseldorf , germany ) , and emission was detected with a monochrome digital camera ( ds - qi1mc ; nikon instruments , dsseldorf , germany ) . images were collected with either a 20x/0.75 ( planapovc , nikon instruments , dsseldorf , germany ) air objective or a 60x/1.40 oil immersion objective ( planapovc , nikon instruments , dsseldorf , germany ) . nis - elements software ( nikon instruments , dsseldorf , germany ) was used for image acquisition . for confocal microscopy , an olympus bx51wi microscope coupled to a confocal laser scanning system ( fv300 ) equipped with a multiline argon ( 488 nm ) and a helium - neon laser ( 543 nm , both melles griot , bensheim , germany ) was used . images were collected with either a 20x/0.50 ( umplanfl , olympus , hamburg , germany ) , a 40x/0.80 water immersion ( lumplan , olympus , hamburg , germany ) , or a 60x/1.40 oil immersion objective ( planapovc , nikon instruments , dsseldorf , germany ) . a kalman filter 4 was employed at every scan . simultaneous or sequential scanning of both fluorophores revealed no difference in their staining patterns , indicating absence of cross - excitation or spectral bleed through . the thickness of z - plane sections was 1 m , and the number of optical sections varied depending on the preparation . figures show extended focus images as specified in the figure legends , which were calculated from z - stacks of optical sections using imagej software ( nih , bethesda , usa ) . images were overlaid employing adobe photoshop cs2 ( adobe systems , cologne , germany ) . data were statistically analyzed by student 's t - test employing the procedures implemented in originpro 8 g software ( originlab corporation , northampton , ma , usa ) . each set of experiments was performed on at least three tissue slices obtained from different animals . the aim of the present study was to analyze changes in glutamate transporter expression and function in astrocytes in response to a mechanical injury . to this end , we prepared organotypic tissue slice cultures of the mouse hippocampus , a well - established model system in which the layering and basic cellular organization of the neural network are maintained over time . after 1925 days in culture , organotypic tissue slices had flattened to a thickness of 4050 m and were composed of 3 to 4 cell layers . at this stage , propidium iodide assays detected only few dead cells dispersed throughout the slices ( 3 0.6 cells per ca1 subfield ; 18 3.4 in entire preparation ; n = 6 ; data not shown ) , indicating that the preparation was viable and in a stable condition . to visualize astrocytes in cultured slices , we used hippocampi of transgenic mice in which green fluorescent protein ( gfp ) is expressed under the control of the promoter of the astrocyte - specific intermediate filament glial fibrillary acidic protein ( gfap ; fvb / n - tg[gfapgfp ] ; gfp / gfap mice ) . a visualization of the gfp fluorescence demonstrated that the general distribution and organization of astrocytes were preserved in the strata pyramidale and radiatum of the ca1 region of organotypic cultures , confirming earlier reports ( n = 3 ; figure 1(a ) ; [ 44 , 45 ] ) . the vast majority of gfp - expressing cells ( ~95% ) also labeled with the astrocyte - specific vital marker sr101 in organotypic slices ( n = 3 ; figures 1(a ) and 1(b ) ) . furthermore , immunohistochemical stainings for gfap in slice cultures derived from wildtype animals showed that the majority of gfap - positive cells were also positive for s100 ( n = 12 ; figure 1(c ) ) , a marker for mature astrocytes . taken together , the nearly complete overlap in the cellular staining pattern for the vital dye sr101 with the expression of gfp in gfp / gfap mice , as well as the overlap in the immunofluorescence for gfap and s100 in organotypic slices indicates that these markers are well suited to identify astrocytes in this preparation . to induce astrogliosis in organotypic slice cultures , we used a scalpel blade and performed a scratch under semisterile conditions through the entire ca1 region oriented perpendicular to the stratum pyramidale ( figure 2(a ) ) . 6 - 7 days after the lesion , a general enhancement of gfap immunoreactivity was observed , indicating astrogliosis ( figure 2(b ) ) . the greatest enhancement of gfap immunoreactivity occurred within a distance of less than 100 m along the lesion ( n = 26 ; figures 2(b ) and 2(c ) ) . here , a dense meshwork of thick , gfap - positive labels was present , indicative of the formation of a glial scar . furthermore , the region close to the lesion site exhibited a discernible increase in s100 immunoreactivity ( n = 12 ; figure 2(c ) ) . both the prominent expressions of gfap and s100 are characteristics of strongly reactive astrocytes , which represent the major cellular component of the glial scar forming after mechanical tissue injury . more distal to the lesion ( > 100350 m ) , a less dramatic rearrangement of gfap immunoreactivity was observed . a clear difference in astrocyte properties between the scar region and its periphery was also seen following sr101 labeling . in the periphery of the scar ( 100350 m distance from the lesion ) , sr101 resulted in a reliable and bright cellular staining , similar to what had been observed in control , unlesioned slices ( n = 12 ; figure 3(a ) , cf . figure 1 ) . in contrast to this , cellular sr101 staining was detectable but only faint within a distance of less than 100 m on both sides of the lesion ( figure 3(a ) ; mean width of sr101-free area 111 3 m ; n = 5 ) . in slices from gfp / gfap transgenic animals ( n = 5 ; figure 3(b ) ) , gfap - positive , weakly sr101-positive scar cells also stained with the sodium - sensitive fluorescence indicator sbfi - am ( acetoxymethyl ester of sodium binding benzofuran isophthalate ; see below ; n = 3 ) , indicating that they were able to take up and de - esterify this dye ( figure 3(b ) ) . in addition to gfap - positive cells , the scar region hosted microglial cells , identified by their vital staining with texas red - coupled lectin from lycopersicon esculentum ( tomato lectin ; n = 4 ; not shown ) . tomato lectin - positive microglia , however , did not take up sbfi - am ( n = 4 ; not shown ) , confirming earlier reports that microglial cells do not incorporate am - ester dyes in situ . to further characterize reactive gliosis in response to the scratch wound , we determined the area of astrocyte somata in confocal images of the gfp fluorescence in control and lesioned slices obtained from gfp / gfap transgenic animals . these measurements revealed that , compared to astrocytes in control slices , the soma area of astrocytes in the periphery of a lesion was increased by 18% and that of astrocytes in the scar region was increased by 24% ( control : 163 2 m ; lesion periphery : 193 3 m ; scar tissue : 201 4 m ; p < 0.001 ; n = 945 , 496 , and 439 in 7 control and 9 lesioned slices ; figures 4(a ) and 4(b ) ) . thus , astrocytes show a second hallmark of reactive gliosis after lesion , namely , a hypertrophy of their cell bodies , which is observed both in the scar tissue as well as in the periphery of the lesion . taken together , these data show that 6 - 7 days after the mechanical lesion , a glial scar has formed along the lesion site . this scar encompasses strongly activated astrocytes , characterized by a robust increase in gfap and s100 expression . these proximal reactive astrocytes , located within a distance of less than 100 m from the lesion , exhibit long gfap - positive processes , have significantly swollen somata , and only weakly stain with sr101 . astrocytes in the periphery of the scar ( 100350 m from the lesion ) display swollen cell bodies as well , but show only moderate upregulation of gfap and maintain their ability to efficiently take up sr101 . to determine the spatial distribution of the glutamate transporters glast and glt-1 in organotypic slices and changes therein in response to the lesion , immunohistochemistry was employed . in control slices , glast ( n = 13 ; figure 5(a ) ) as well as glt-1 immunoreactivity ( n = 11 ; figure 5(b ) ) appeared as punctate labeling throughout the entire ca1 area . the staining patterns for both transporters in the tissue slice cultures were thus similar to those reported from acute tissue slice preparations of the hippocampus [ 36 , 48 , 49 ] . the relatively homogeneous distribution of immunoreactivity throughout the preparation found in control slice cultures was abandoned following a lesion . here , distinct accumulations of glast ( n = 17 ; figure 5(a ) ) and glt-1 immunoreactivity ( n = 12 ; figure 5(b ) ) were found in the scar region . in both cases , these preferentially colocalized with thick gfap - positive bundles running in parallel to the lesion ( figure 5 ) . apart from the marked accumulation of glast reactivity in parallel to the scar , glast immunoreactivity appeared generally increased in some lesioned slice preparations ( figure 5(a ) ) but unaltered in others both within the scar as well as in its periphery . for glt-1 , a decline in the intensity of immunoreactivity was observed with increasing distance from the lesion ( figure 5(b ) ) . in summary , these results reveal distinct alterations in the immunoreactivity of glast and glt-1 following a mechanical lesion . in the scar region , clusters of glt-1 and glast immunoreactivity are present , primarily colocalized with strongly gfap - positive fibers . apart from these distinct clusters , glt-1 immunoreactivity appears to decline with distance from the scar , whereas glast immunoreactivity seems largely uniform throughout the preparation . glutamate uptake is accompanied by the inward flux of sodium , resulting in increase in the intracellular sodium concentration of astrocytes . to monitor its function , we assayed sodium increases induced by d - aspartate , a nonmetabolized substrate of sodium - dependent glutamate uptake which is also transported in astrocytes in the stratum radiatum of organotypic slices . to this end , sr101-stained slices were additionally loaded with the sodium - sensitive fluorophore sbfi - am ( figure 6(a ) , cf . the sbfi fluorescence revealed a linear increase in its fluorescence ratio at intracellular sodium concentrations between 10 and 40 mm ( n = 107 ; figure 6(b ) ) as reported before . repetitive pressure application of d - aspartate for 500 ms induced reliable increases in the intracellular sodium concentration in astrocytes , indicating activation of sodium - dependent glutamate uptake ( n = 488 ; figure 6(c ) ) . the experiments were performed in the presence of the sodium - channel blocker ttx ( tetrodotoxin , 0.5 m ) to suppress action potential generation . the amplitude of d - aspartate - induced sodium increases was dependent on the concentration applied ( figure 6(c ) ) and saturated at about 2 mm ( n = 19 ; figure 6(d ) ) , as reported before for mouse cortical astrocytes in culture . at a concentration of 1 mm , the amplitude of d - aspartate - induced sodium transients was near maximum and amounted to 3.8 0.2 mm ( n = 161 ; figures 6(c ) and 6(d ) ) , a value comparable to that in astrocytes in acute slices obtained in our lab . to probe for changes in glutamate uptake , we compared sodium signals induced by 1 mm d - aspartate between control and lesioned slices ( figure 7 ) . in lesioned slices , astrocytes located in the periphery of the lesion ( 100350 m distance ) responded with a sodium transient to application of 1 mm d - aspartate ( see cell 2 in figures 7(a ) and 7(b ) ) . the amplitude of d - aspartate - induced sodium signals in cells in the lesion periphery was similar to that observed in control slices ( 3.8 0.3 mm ; n = 111 ; see cell 1 in figures 7(a ) , 7(b ) , and 7(c ) ) . application of d - aspartate also evoked sodium transients in weakly sr101-positive , sbfi - filled astrocytes located in the glial scar area ( < 100 m from the lesion ; see cells 35 in figures 7(a ) and 7(b ) ) . these cells , however , displayed significantly smaller peak amplitudes ( 1.6 0.21 mm , n = 26 ; p < 0.001 ; figures 7(b ) and 7(c ) ) than astrocytes in the periphery and in control slices . the kinetics of d - aspartate - induced sodium transients differed between all three groups . this was true for the slope of the sodium increase ( 10%90% change ) , which was significantly reduced in astrocytes from lesioned slices versus astrocytes from control slices ( 0.9 0.05 mm / s in control versus 0.6 0.06 mm / s in the periphery and 0.2 0.04 mm / s in the scar region ; n = 161 , 111 , and 26 , resp . ; in addition , the decay back to baseline was significantly slower following lesion ( figures 7(b ) and 7(c ) ) . the time needed for the signal to decay by 67% was approximately doubled ( decay time was 44 2 s in control , = 81 4 s in periphery of the lesion , and = 95 10 s in scar cells ; figures 7(b ) and 7(c ) ) . these results show that mechanical injury results in distinct alterations in the kinetics and/or amplitudes of d - aspartate - induced intracellular sodium transients in reactive astrocytes , depending on their distance to the lesion . our results show that within 6 - 7 days after setting a scratch wound in organotypic hippocampal slices , a dense glial scar had formed along the lesion site . up to approximately 100 m distance from the lesion , the scar tissue was characterized by a substantial elevation of gfap expression , a pronounced cellular hypertrophy , and reduced uptake of sr101 by astrocytes . while prominent clusters of glast and glt-1 immunoreactivity were observed along gfap - positive structures in scar cells , d - aspartate - induced intracellular sodium signals were strongly dampened , indicating a significant reduction in glutamate uptake capacity close to the lesion . in the periphery of the lesion ( 100350 m ) , astrocytes showed less pronounced reactivity , maintained their ability to efficiently take up sr101 , and displayed only minor changes in glutamate transporter immunoreactivity and function . thus , depending on their distance from the lesion , astrocytes showed different grades of reactivity and displayed discrete changes in glutamate transporter immunoreactivity and uptake capacity . organotypic tissue slice cultures of the hippocampus represent a well - established model system , in which the basic tissue architecture is maintained , but which still hosts the major advantages of cell cultures such as good accessibility and control of experimental conditions . they have been used extensively for the analysis of neuronal properties and developing neuronal networks [ 28 , 51 , 52 ] or the study of excitotoxic neuronal damage ( e.g. , [ 53 , 54 ] ) . recent studies also demonstrated that basic morphological characteristics of astrocytes and the typical glia - synapse organization are well preserved in organotypic slice cultures ( e.g. , [ 44 , 45 , 55 , 56 ] ) . in addition , we found a nearly complete overlap of sr101 staining with the expression of gfp in slice cultures obtained from gfp / gfap mice , demonstrating that sr101 is well suited to identify astrocytes in this preparation as described before for acute tissue slices and in vivo [ 29 , 30 ] . performing a scratch through the ca1 region of organotypic slices resulted in the formation of a glial scar along the lesion site , as judged by the prominent increase in expression of gfap and s100. in addition , astrocyte cell bodies showed a significant hypertrophy . furthermore , astrocytes in the periphery of the scar ( 100350 m from the lesion ) showed mild to moderate reactivity , displaying swollen cell bodies , and only moderate upregulation of gfap . these changes are hallmarks of reactive gliosis observed after tissue injury [ 10 , 13 , 57 ] . generally , the degree of injury and the distance of the astrocytes from the site of injury define the degree of their activation [ 11 , 13 ] . our study also revealed that strongly reactive astrocytes in the scar region only weakly stain with sr101 . the reduced ability of strongly activated astrocytes to accumulate sr101 indicates that strong glial activation is accompanied by a downregulation of the organic anion transporter responsible for uptake of sr101 . this is reminiscent of immature astrocytes in the early postnatal hippocampus , which are sr101-negative and in line with a wealth of experimental evidence suggesting that reactive gliosis represents a process comprising a dedifferentiation of astrocytes . glutamate uptake in the hippocampus is mainly achieved by the glial glutamate transporters glt-1 and glast [ 1 , 2 ] . a central factor in many brain pathologies , including traumatic brain injury , is an increase in extracellular glutamate and excitotoxicity ; it has been suggested that a change in the expression levels of glial glutamate transporters might play a critical role in the failure of glutamate clearance [ 6 , 5860 ] . earlier work has found an overall downregulation of protein levels of both glast and glt-1 following astrocyte activation [ 2227 ] , indicating that this is causal to the elevation of extracellular glutamate . in contrast to this notion , other studies reported increased glutamate transport capacity of reactive astrocytes and suggested a protective influence [ 61 , 62 ] . to visualize possible differences in the spatial expression profile of glt-1 and glast in strongly reactive astrocytes along the scar as compared to moderately reactive astrocytes in its periphery , we performed immunohistochemical stainings . these revealed an accumulation of glt-1 and glast immunoreactivity along thick gfap - positive fibers , which was particularly pronounced for glast . besides these clusters , overall glast immunoreactivity seemed unaltered as compared to unlesioned slices , whereas glt-1 immunoreactivity seemed weaker and declined with increasing distance from the scar . clustering of glutamate transporters has been described by several reports [ 48 , 63 , 64 ] . in developing hippocampal astrocytes , cluster formation was preferentially found in branches opposed to synapses and was increased with increased neuronal activity , indicating that it is necessary to cope with synaptic release of glutamate . along the same lines , it was observed that sustained astroglial activation by ciliary neurotrophic factor ( cntf ) in the rat striatum induced a concentration of glast and glt-1 into raft microdomains and improved glutamate clearance , indicating that cluster formation increased the cellular capacity for glutamate uptake . however , another study found glutamate - induced clustering of glt-1 that induced its endocytosis and intracellular trafficking without changing the total expression levels as detected by western blots , arguing for a decrease in functional glial glutamate uptake capacity . in the present study , antibodies against glt-1 and glast were employed for immunohistochemistry after permeabilization of the plasma membrane . the clusters of glast and glt-1 along gfap - positive fibers in the scar region ( as glast and glt-1 immunoreactivity in general ) could thus represent glutamate transporters in the plasma membrane or in intracellular compartments or both . consequently , the observed clustering does not allow a prediction about possible functional changes in glutamate uptake . we probed for the functional activation of glutamate transport by application of the transportable agonist d - aspartate , which results in an increase in the intracellular sodium concentration of astrocytes [ 8 , 9 ] . the amplitudes of d - aspartate - induced sodium transients in astrocytes in control organotypic slices were similar to those reported from astrocytes in acute slices obtained in our lab , indicating similar cellular glutamate transport capacity in both preparations . following a lesion , d - aspartate - induced intracellular sodium signals were clearly dampened in scar cells . moreover , the slope of the increase in sodium was significantly reduced , and the decay back to baseline was slowed . because d - aspartate was applied at nearly saturating concentration , the reduction in peak amplitudes suggests a reduction in functional glutamate uptake capacity in cells close to the lesion , which might be mediated by a decrease in the overall number of glutamate transporters available at the plasma membrane . in contrast , the alteration in the kinetics of the d - aspartate - induced sodium signals can be explained by the cellular hypertrophy that was observed in reactive astrocytes . at an equal transport - mediated sodium influx across the membrane , the slope of resulting changes in the sodium concentration will be decreased in cells with a larger volume . furthermore , the decay of intracellular sodium transients back to baseline is mainly governed by the activity of the na / k - atpase as well as by diffusion . the slower recovery is in line with earlier studies reporting a downregulation of the sodium pump following reactive gliosis [ 65 , 66 ] . moreover , sodium is not buffered in the cell and travels solely by means of diffusion in the cytoplasm . hindered diffusion , resulting from increased cytoplasmic protein content or impaired gap junction coupling which both occur upon astrocytic activation , might thus also partially contribute to delayed sodium recovery [ 12 , 34 , 67 ] . in contrast to scar cells , the amplitude of d - aspartate - induced intracellular sodium signals was not significantly altered in moderately activated astrocytes in the periphery of the lesion , indicating that the overall number of functional glutamate transporters was not significantly altered . because these cells showed hypertrophy as well , the slowed kinetics of the sodium transients are in line with an increased cellular volume as well as a possible downregulation of na / k - atpase and/or a slowed diffusion as argued above . taken together , our results indicate a significant reduction in glutamate uptake capacity in strongly activated astrocytes in the scar region . thus , the prominent clustering of glast and glt-1 immunoreactivity along gfap - positive structures close to the lesion is likely to reflect a loss of glutamate transporters from the plasma membrane upon transporter internalization as reported by nakagawa et al . . the authors of the latter study speculated that large and prolonged increases in glutamate concentrations are necessary to induce such cluster formation and endocytosis in the tissue , as they would possibly only occur under pathological conditions . indeed , ambient glutamate concentrations were shown to be significantly increased after traumatic brain injury [ 6870 ] . along these lines , it can be assumed that in our model system , extracellular glutamate concentrations rose less severely in the periphery of the lesion , preventing a comparable clustering and loss of functional glutamate transport activity in moderately activated astrocytes . our results show complex changes in glutamate transporter expression and function during astrocyte activation following mechanical injury . they confirm that the degree of astrocyte activation depends on the distance from the insult and comprises different endpoints regarding cellular morphology and physiology . furthermore , our results highlight that astrocytes which show different grades of reactivity also display discrete changes in glutamate transporter expression and function . while immunohistochemistry revealed a prominent clustering of glt-1 and glast immunoreactivity in the scar region , our functional assay clearly showed that glutamate uptake capacity is strongly reduced in scar cells , while it is largely maintained in moderately activated astrocytes in the periphery . thus , mild - to - moderate astrogliosis in the periphery of a mechanical lesion does not necessarily seem to be accompanied by a significant change in glial glutamate uptake capacity . at the glial scar itself , in strongly reactive astrocytes , a clustering of glutamate transporters is observed that apparently goes along with a severe functional reduction in astroglial glutamate uptake , which may contribute to glutamate - mediated excitotoxicity in this region .

astrocytes express the sodium - dependent glutamate transporters glast and glt-1 , which are critical to maintain low extracellular glutamate concentrations . here , we analyzed changes in their expression and function following a mechanical lesion in the ca1 area of organotypic hippocampal slices . 6 - 7 days after lesion , a glial scar had formed along the injury site , containing strongly activated astrocytes with increased gfap and s100 immunoreactivity , enlarged somata , and reduced capability for uptake of sr101 . astrocytes in the scar 's periphery were swollen as well , but showed only moderate upregulation of gfap and s100 and efficiently took up sr101 . in the scar , clusters of glt-1 and glast immunoreactivity colocalized with gfap - positive fibers . apart from these , glt-1 immunoreactivity declined with increasing distance from the scar , whereas glast expression appeared largely uniform . sodium imaging in reactive astrocytes indicated that glutamate uptake was strongly reduced in the scar but maintained in the periphery . our results thus show that moderately reactive astrocytes in the lesion periphery maintain overall glutamate transporter expression and function . strongly reactive astrocytes in the scar , however , display clusters of glast and glt-1 immunoreactivity together with reduced glutamate transport activity . this reduction might contribute to increased extracellular glutamate concentrations and promote excitotoxic cell damage at the lesion site .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

PMC4327690

wear resistance of denture teeth has been considered as one of the most important requirements for oral rehabilitation of edentulous patients with removable dentures , in order to maintain a stable occlusal support over time13 . wear of the occlusal surfaces may result in insufficient posterior tooth support25 , loss of chewing efficiency1,21 and nonfunctional activities1,2 . although wear of acrylic resin teeth has also been related to the loss of vertical dimension of occlusion with complete dentures , the major factor affecting it is the reduction of residual ridges by resorption22 . initially , denture teeth were made of ceramic material . with the advent of polymethyl methacrylate ( pmma ) in the 1940s , denture teeth are currently made of either methacrylate - based resins ( acrylic resin ) or ceramics , although resin teeth have almost eliminated ceramic teeth from the market25 due to a number of advantages , such as the chemical bond to denture base5,6,16 , lower susceptibility to fracture6 and decrease of clicking4,24 . nonetheless , the wear resistance of acrylic resin teeth has been questioned for being lower than that of ceramic teeth10 - 12,18,20,22,28 . manufacturers have then tried to develop acrylic resins designed to offer improved wear resistance for resin denture teeth25 . pmma has been reported to have advanced with the advent of cross - linked agents27 , which are bifunctional monomeric molecules that are added to the polymeric material to allow crossing between linear polymeric chains11 . the cross - linked polymer was later modified by increasing the quantity of cross - linked agents to ensure a higher degree of cross - linking between the polymers22 , or by blending special polymers and co - polymers.3 other modification of cross - linked polymer was the appearance of resins with interpenetrating polymer network ( ipn ) 22,28 . ipn resin is formed when a polymer network is crossed inside another network occupied by a second crossed polymer . the crossed networks coexist in the same volume of the space ( one being physically retained within the other ) and can not be separated without chemical bond rupture22,29 . these factors may influence the final quality for the acrylic resin and produce denture teeth with better mechanical and physical properties26 . the search for a more wear - resistant denture tooth material resulted in the development of modified resin teeth that display acceptable wear resistance21 . in present days , acrylic resin denture teeth can be classified into two main categories : conventional ( with crossed - linked polymeric chains ) and high - strength ( with various polymer technologies that provide different chemical compositions ) . some studies22,27,28 have found a higher wear resistance for the high - strength acrylic resin denture teeth with modified polymers in comparison to conventional pmma , while others14,15,17,19,29 have found no advantages of such materials with regard to wear properties . composite resin denture teeth were developed in the 1980s as an effort to achieve greater wear resistance and bond strength to denture bases30 . tooth materials made with microparticle inorganic fillers immersed in a bis - gma ( bisphenol a glycidyl methacrylate ) matrix1,28 , or nanometric inorganic fillers immersed in a pmma matrix26 have been used for fabrication of composite denture teeth . it has been reported that composite denture teeth show a higher wear resistance than acrylic resin denture teeth3,26 , although data vary depending on the experimental design3 . moreover , some authors found denture teeth with inorganic fillers to be 40 - 50% more abrasion - resistant than ipn teeth27 . despite the evolution of the mechanical properties of pmma , wear of this material continues to occur with clinical use10,12 . the wear resistance of acrylic resin denture teeth has been investigated22,27,28 ; however , the constant appearance of resins with modified polymers used for fabrication of denture teeth manufacturing , impose the need for further studies in this context . furthermore , the results are not clear because the wide variety of wear - testing devices have hindered the comparison of the results19,26 , which reflect the lack of a standardized and reproducible methodology for testing the wear resistance of acrylic resin denture teeth28 . this in vitro study tested the null hypotheses that there is no difference among the wear resistance of one conventional ( trubyte biotone ) and two high - strength pmma denture teeth ( trilux and vivodent ) when opposed to glazed or airborne particle abraded ceramic . the two different antagonist ceramic surfaces were used to allow the test to be carried out under conditions that would cause minimal and maximal wear of the denture teeth . both right and left maxillary canines ( n=10 ) of 3 types of pmma denture teeth were purchased : trubyte biotone ( a conventional denture tooth composed by cross - linked pmma ) ; trilux ( a high - strength tooth composed by highly cross - linked ipn - pmma ) ; and vivodent ( a high - strength tooth composed by highly cross - linked pmma ) ( table 1 ) . all teeth were stored in distilled water at 37c for 24 h before wear testing to allow water absorption . * the in vitro two - body wear - testing apparatus used in this study was a simulated brushing machine . the denture tooth was fixed on the top of a tooth brush using a screw and composite resin to allow the tooth be fixed on the wear - testing apparatus . the tooth was securely mounted on the upper stainless steel holder of the apparatus ( movable part ) , whereas the antagonist plate was mounted on the fixed bottom part so that they contact tightly on each surface ( figure 1 ) . glazed or airborne particle abraded metalloceramic plates ( 2.5 x 1.2 x 0.15 cm ) were used as antagonist surfaces and replaced after each change of denture tooth . the opposing plates were cast in ni - cr alloy ( durabond universal , odonto comercial importadora ltda ) and trimmed with aluminum oxide abrasive burs at low rotation . thereafter the surface was sandblasted with 100 m aluminum oxide particles ( trijato odonto larcon , maring , pr , brazil ) and the conventional application of porcelain was made , including the glaze final process ( duceram plus , degudent ind . the antagonist ceramic was planed and polished using silicon carbide paper up to 600 ( extec corp . , enfield , usa ) and then sandblasted with 100 m aluminum oxide particles . at each change of paper , the metalloceramic plates were cleaned in an ultrasonic cleaner at 40 hz for 2 min . wear testing was performed by repeated sliding contact at 4.5 cycles / s with 20 mm sliding distance per cycle in the buccolingual direction with a load of 300 g. during the wear test , continuous rinsing with demineralized water ( 37c ) was used to remove abraded particles from the sample surface and to simulate the wet environment of the oral cavity . the height ( mm ) of each tooth was measured using a digital measuring microscope accurate to 0.001 mm ( tm-505 , mitutoyo corporation , japan ) before and after the wear test . the wear resistance assessed as height loss ( mm ) was calculated as the decrease in height after 100,000 cycles . the mean wear values were analyzed individually by two - way analysis of variance ( anova ) and the differences between the denture teeth were determined by the tukey 's test ( =0.05 ) . the mean wear values obtained after 100,000 cycles of all denture teeth tested against the glazed or airborne particle abraded ceramic plates are summarized in table 2 . there were no statistically significant differences ( p>0.05 ) in wear among the 3 denture teeth evaluated against glazed ceramic . there were significant differences among the teeth tested against abraded ceramic , with trubyte biotone presenting significantly more wear compared to the high - strength denture teeth ( p<0.0001 ) , which , in turn , did not differ significantly from each other ( p>0.05 ) when tested against either glazed or airborne particle abraded ceramic . all teeth showed significantly more wear against airborne particle abraded than against glazed ceramic ( p<0.05 ) . * data with the same letters are not statistically different within the same antagonist ceramic at 5% significance level . a large number of chewing simulators serving to determine the in vitro wear of acrylic resin denture teeth have been described8,24,30 . in vitro methods three - body wear tests are frequently used to measure wear characteristics . in these tests , in addition to the antagonists , various factors are taken into consideration , namely the abrasive properties of the intermediate medium or ph - related dissolution processes . on the other hand , in two - body wear tests , all substance losses result from a direct interaction between the specimen surface and the antagonist surface31 . clinically , this situation arises , for example , during swallowing , empty mastication , parafunctional habits and dynamic occlusion movements . especially in the case of full dentures with bilaterally balanced occlusion , these direct occlusal contacts are a factor that contributes to the wear of denture teeth . therefore , a two - body wear simulation was performed in the present this study25 . the wear - testing apparatus used in this study was developed to evaluate two - body wear of the pmma teeth abraded directly against glazed or airbone particle abraded opposing ceramic without an intermediate abrasive medium . the obtained data showed that the wear of resin denture teeth was influenced considerably by the texture of the opposing material . the least wear indicated as the total height loss was observed with trilux tooth opposing the glazed ceramic . however , a very small amount of wear occurred in all denture teeth tested against glazed ceramic , which suggests that the difference on wear among the teeth could not have been detected in this way . indeed , no significant differences were observed on the wear of the teeth tested against the glazed ceramic ( p>0.05 ) . nonetheless , with regard to the data obtained from the airborne particle abraded ceramic , trubyte biotone ( a conventional denture tooth made of cross - linked pmma ) showed significantly more wear than trilux ( a high - strength denture tooth made of highly cross - linked ipn - pmma ) and vivodent ( a high - strength denture tooth made of highly cross - linked pmma ) . since higher mean wear values were found against the abraded ceramic compared to the glazed ceramic , these results appear to be more consistent in this current wear test series . it is thus assumed that a connection between the chemical composition and the wear resistance of these denture teeth may rather exist . these data are consistent with those other studies that have also found a lower wear resistance for conventional pmma denture teeth evaluated in comparison to high - strength denture teeth8,21,24,28,27,26 . however , regarding the high - strength teeth evaluated in the present study ( trilux and vivodent ) , no significant differences were found in wear for either of the opposing ceramics . this finding was in accordance with some authors15,19,21 and had previously been reported by other study3 . in spite of that , differences in the wear of high - strength denture teeth formulations have been reported27 . therefore , the results of previous in vitro studies on wear resistance of acrylic resin denture teeth seem to be rather inconsistent25 . the findings of whitman , et al.28 , hirano , et al.13 , and suzuki26 have suggested that denture teeth made of ipn resin , highly cross - linked polymers or composite resin are more wear - resistant than the conventional acrylic resin denture teeth made of cross - linked pmma . 25 , on the other hand , did not find any connection between the chemical composition and the wear resistance of the tested denture teeth . other investigations also failed to show differences among denture teeth made of polymers with a high degree of cross - linking or polymers with inorganic fillers and teeth made of conventional pmma3,14,15,19,22,28,29 . these evidences put in question the superiority of high - strength denture teeth and raises doubts about the advantageous clinical use of these products over time . indeed , recent clinical investigations have not revealed any statistically proven difference in wear behavior between polymers with a high degree of cross - linking or with inorganic fillers and conventional polymethyl methacrylate15,19,22 . these conflicting data may be due to the large variety of experimental designs , measuring instruments and wear - testing methods used in these investigations . the large number of denture tooth brands with different chemical compositions has created additional difficulties to the analysis of these data . in spite of the controversy the results of this study may assist dentists in selecting pmma denture teeth from the standpoint of wear resistance . nonetheless , it is important to consider that there is not a determinant factor for predicting the wear of pmma denture teeth . on the contrary , besides the chemical composition of the acrylic resin , several other factors should be taken into account on the abrasive process to allow the scientific understanding of the complex phenomenon of wear , namely the chewing pattern10,18 , chewing frequency14 , occlusion force , food abrasion10,18 , non - functional tooth - grinding habits11 , abrasive cleansers3 , materials ' mechanical properties1,5,20 and dusty atmosphere12 . under the experimental conditions and within limitations of this study , the null hypothesis was rejected , since the wear resistance of the three types of polymethyl methacrylate denture teeth was different against glazed or airborne particle abraded ceramic .

the wear resistance of denture teeth is important to the longevity of removable prostheses of edentulous patients . the ability of denture teeth to maintain a stable occlusal relationship over time may be influenced by this property . the purpose of this in vitro study was to evaluate the wear resistance of polymethyl methacrylate ( pmma ) denture teeth based on their chemical composition when opposed by a ceramic antagonist . the maxillary canines ( n=10 ) of 3 pmma denture teeth ( trubyte biotone , cross - linked pmma ; trilux , highly cross - linked ipn ( interpenetrating polymer network)-pmma ; and vivodent , highly cross - linked pmma ) were secured in an in vitro 2-body wear - testing apparatus that produced sliding contact of the specimens ( 4.5 cycles / s , sliding distance of 20 mm , under 37c running water ) against glazed or airborne particle abraded ceramic . wear resistance was measured as height loss ( mm ) under 300 g ( sliding force ) after 100,000 cycles , using a digital measuring microscope . mean values were analyzed by 2-way anova and tukey 's test ( =0.05 ) . the wear of trubyte biotone ( 0.93 0.14 mm ) was significantly higher than that of both other types of teeth tested against abraded ceramic ( p<0.05 ) . the vivodent tooth ( 0.64 0.17 mm ) exhibited the best wear resistance among the denture teeth tested against airborne particle abraded ceramic . there were no statistically significant differences ( p>0.05 ) in wear among the 3 denture teeth evaluated against glazed ceramic . trilux and vivodent teeth tested against either glazed or airborne particle abraded ceramic did not differ significantly from each other ( p<0.05 ) . all teeth showed significantly more wear against airborne particle abraded ceramic than against glazed ceramic ( p<0.05 ) . in conclusion , the three types of pmma denture teeth presented significantly different wear resistance against the abraded ceramic . the high - strength pmma denture teeth were more wear - resistant than the conventional pmma denture tooth .

INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION CONCLUSIONS

PMC4616120

humans can lead efficient daily lives by adjusting their walking patterns in various ways during exercise . also , general walking does not efficiently control calories , the heart rate , the oxygen consumption , and the respiratory exchange ratio during the activity2 . however , nordic walking activates the upper limb muscles and lowers the efficiency of recruitment and weight - bearing of the lower limbs , as the duration of the fatigue lengthens3,4,5 . the primary purpose of using a pole is to be able to use the upper limb muscles , which are not normally used in walking , and to facilitate high - intensity exercise with the minimum effort by adjusting the energy consumption of the body6 . according to the research of schwameder , nordic walking increased the strengths of the upper and lower limb muscles , and the poles dispersed the body weight ; furthermore , it was found to lower the vertical ground reaction force of the knee and significantly change the knee extension angle7 . therefore , the present study analyzed the effects of nordic walking and walking on spatiotemporal parameters and ground reaction force , so that it would be possible to suggest an efficient means of walking for people who walk for health . the subjects were divided into a nordic walking group of 15 subjects and a walking group of 15 subjects . all subjects signed an informed consent form approved by the sehan university institutional review board . the general characteristics of the subjects were measured using an inbody j05 system ( biospace , usa ) . the nordic walking group s average age was 23.2 4.6 years , average height was 167.2 4.3 cm , and average weight was 63.4 4.7 kg . the walking group s average age was 23.8 3.9 years , average height was 169.4 5.2 cm , and average weight was 65.3 5.78 kg . the three - dimensional kinematic and kinetic changes in the joints while walking were analyzed using a six - camera vicon mx motion analysis system ( oxford metrics group , oxford , uk ) for the gait test of the subjects . markers were placed on each of the subjects lower limbs in the standard places ( table 1table 1.marker locationmarker numberlocation1 and 2bilateral psis3 and 4bilateral asis5 and 6midway between the hip and knee , lateral surface of the thigh7 and 8lateral femoral epicondyle9 and 10midway between the knee and ankle , lateral surface of the shank11 and 12lateral malleolus13 and 14calcaneal tuberosity15 and 16proximal to the second metatarsal headasis : anterior superior iliac spine ; psis : posterior superior iliac spine ) . for analysis of gait , values of cadence ( step / min ) , stride time ( seconds ) , step time ( seconds ) , step length ( mm ) , stride length ( mm ) , and ground reaction force were analyzed . a static test was conducted on the force plate in a stationary standing posture . for a dynamic test , the subjects were asked to walk 12 meters using the walking method more comfortable for them , that is , nordic walking or walking ; after they walked 12 meters more than 10 times , their most natural walking patterns were chosen three times and analyzed . to determine the pole length for nordic walking , each subject s height asis : anterior superior iliac spine ; psis : posterior superior iliac spine the statistical analysis was carried out using a commercial statistics program , pasw statistics for windows version 18.0 ( spss , chicago , il , usa ) . all the data on the measured items were expressed as averages and standard deviations ; to verify the differences in spatiotemporal parameters and ground reaction force , a paired t - test was performed . to verify the statistical significance of each result , comparison of the spatiotemporal gait parameters showed that cadence ( 118.845.95 steps / min vs. 104.106.32 steps / min ) , stride length ( 1.420.10 m vs. 1.300.15 m ) , and step length ( 0.720.06 m vs. 0.700.08 m ) were increased and stride time ( 1.010.05 sec vs. 1.16 0.07 sec ) and step time ( 0.510.03 sec vs. 0.590.04 sec ) were decreased for nordic walking compared with walking , respecively ( table 2table 2.comparison of spatiotemporal parameters for walking and nordic walkingcharacteristicswalkingnordic walkingcadence ( steps / min)104.106.32118.845.95***stride time ( s)1.16 0.071.010.05***step time ( s)0.590.040.510.03***stride length ( % ll)1.300.151.420.10**step length ( % ll)0.700.080.720.06*values are shown as the meansd . * p<0.05 ; * * p<0.01 ; * * * p<0.001 ) . values are shown as the meansd . * p<0.05 ; * * p<0.01 ; * * * p<0.001 in the initial stance phase ( fz1 ) , walking and nordic walking showed ground reaction forces of 105.109.26% body weight ( bw ) and 117.8512.70% bw , which were significantly different ( p < 0.01 ) . in the mid - stance phase ( fx0 ) , walking and nordic walking showed ground reaction forces of 74.277.23% bw and 66.1510.10% bw , which were significantly different ( p < 0.01 ) . in the terminal stance phase ( fx2 ) , walking and nordic walking showed ground reaction forces of 110.205.27% bw and 114.407.06% bw , which were significantly different ( p < 0.05 ) ( table 3table 3.comparison of vertical ground reaction force for walking and nordic walkingwalkingnordic walkingfz1 max105.109.26117.8512.70**fz0 max74.277.2366.1510.10**fz2 max110.205.27114.407.06*values are shown as the meansd . this study was conducted to identify the effects of nordic walking and walking on spatiotemporal parameters and ground reaction force , so that it would be possible to suggest an efficient means of walking for people who perform nordic walking for health . significant differences were in the spatiotemporal gait parameters between nordic walking and walking . in nordic walking , cadence , stride length , and step length were increased compared with walking , but stride time and step time were decreased . they reported that the spatiotemporal parameters were remarkably increased during walking , the time of contact with the ground in nordic walking was decreased , and cadence , step length , and stride length were obviously increased8 . in the present study , it is thought that weight dispersion decreased energy consumption and increased the walking speed due to use of the upper limbs in nordic walking , though the propulsive force was adjusted in the lower limb muscles . it is also thought that the calf muscles improved the stability of the ankle joints9 and promoted quick propulsion in the terminal stance phase during nordic walking10 . this study analyzed the changes in vertical ground reaction force in nordic walking and walking . the results showed significant differences in the maximum force of the load reactor in the initial stance phase ( fz1 ) ( p < 0.01 ) and in the terminal stance phase ( fz2 ) ( p < 0.05 ) . this corresponds with the results of previous studies reporting that the vertical ground reaction force ( p < 0.01 ) and weight - bearing of the lower limbs decreased in nordic walking11 , 12 . using an electromyogram , force plate , and motion analysis system , chiu and wang analyzed the perceived exertion , muscle activity , and joint angle of the lower limbs and ground reaction force in 30 adults depending on changes in the velocity of walking . they found that walking speed had a clear effect on the vertical ground reaction force : the vertical reaction force was reduced due to increased walking speed in the mid - stance phase ( fz0 ) , and the maximum force rose in the load reactor in the initial stance phase ( fz1 ) and the terminal stance phase ( fz2 ) . using of nordic walking poles requires an increase in the force of the upper limb muscles to boost the weight - bearing of the body during the stance phase , improves balance and stability , and enhances the metabolism for movement13 . this demonstrates that nordic walking is more effective in improving functional capabilities by promoting effective energy use and reducing the lower limb load , as the weight of the upper and lower limbs is dispersed during nordic walking . to apply nordic walking as an exercise program , it is deemed more important to adjust the natural walking speed and weight - bearing of the body . in conclusion , further kinematic and muscular physiological research should be conducted after subjecting the nordic walking subjects to a long - term training program , and a study on the effect of long - term nordic walking on the elderly or people with a pathologic gait is needed in the future .

[ purpose ] the purpose of this study was to investigate the effects of nordic walking and walking on spatiotemporal gait parameters and ground reaction force . [ subjects ] the subjects of this study were 30 young adult males , who were divided into a nordic walking group of 15 subjects and a walking group of 15 subjects . [ methods ] to analyze the spatiotemporal parameters and ground reaction force during walking in the two groups , the six - camera vicon mx motion analysis system was used . the subjects were asked to walk 12 meters using the more comfortable walking method for them between nordic walking and walking . after they walked 12 meters more than 10 times , their most natural walking patterns were chosen three times and analyzed . to determine the pole for nordic walking , each subject s height was multiplied by 0.68 . we then measured the spatiotemporal gait parameters and ground reaction force . [ results ] compared with the walking group , the nordic walking group showed an increase in cadence , stride length , and step length , and a decrease in stride time , step time , and vertical ground reaction force . [ conclusion ] the results of this study indicate that nordic walking increases the stride and can be considered as helping patients with diseases affecting their gait . this demonstrates that nordic walking is more effective in improving functional capabilities by promoting effective energy use and reducing the lower limb load , because the weight of the upper and lower limbs is dispersed during nordic walking .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC5260547

the role of exercise on adult cognition has been extensively researched ( lambourne and tomporowki , 2010 ; tomporowski , 2003 ) , with cognitive performance assessed during exercise ( mcmorris and graydon , 1996 ) , following an acute bout of exercise ( coles and tomporowski , 2008 ; hopkins et al . , 2012 ; tomporowski et al . , 2005 ) and following long - term exercise exposure ( castelli et al . , 2007 ; hopkins et al . , 2012 ) . despite this , the effect of exercise on cognitive performance is equivocal . this ambivalence is due in part to the different exercise protocols , methods of assessing exercise intensity and mode of exercise employed by various studies ( lambourne and tomporowski , 2010 ) . for example , when investigating the exercise intensity - cognitive performance relationship , several studies and meta - analyses have suggested an inverted u - shaped relationship ( chmura et al . , 1994 ; mcmorris and graydon , 2000 ; mcmorris et al . , 2011 ) such that moderate levels of exercise increased physiological arousal and facilitated cognition , however , when physiological arousal approached a maximal level , cognitive performance began to deteriorate . in contrast , maximal levels of exercise intensity have been found not to be generally associated with declines in cognition ( tomporowski , 2003 ) with research by lyons et al . ( 2008 ) reporting an inverted - u relationship between exercise intensity and post - exercise coincidence anticipation performance in novice performers and not experts ; whereas subsequent work by duncan et al . ( 2012 ) found an inverted - j relationship between exercise intensity and coincidence anticipation performance ( cat ) during treadmill running at 90% of heart rate reserve ( hrr ) , where cat performance incrementally reached an optimal point before dramatically dropping off at 90% of hrr ) . httermann and memmert ( 2014 ) presented data showing an inverted - u relationship in cognitive performance for non - athletes while cycling at exercise intensities of 50 , 60 and 70% of the age predicted maximal heart rate . on the contrary , a linear relationship was seen for athletes , leading the researchers to conclude that physical fitness acted as a moderator in the exercise intensity - cognitive performance relationship ( httermann and memmert , 2014 ) . however , the use of the age predicted heart rate to determine exercise intensity in the httermann and memmert s ( 2014 ) study is limited , particularly when comparing individual fitness levels . for example , regular exercise participation was shown to be associated with a lower resting heart rate ( karvonen et al . , 1957 ) , thus , the use of the age predicted heart rate alone does not accurately account for this issue nor it has any scientific basis in exercise physiology and sports medicine ( robergs and landwehr , 2002 ) where exercise intensity is commonly determined as a percentage of maximal oxygen uptake . furthermore , considering that httermann and memmert s ( 2014 ) high exercise intensity condition was set at 70% of the age predicted maximal heart rate and a linear relationship between intensity and cognitive performances for trained athletes was reported , it suggests that cognitive performance was still on the increase . consequently , it can be proposed that the intensity level of 70% for trained athletes was moderate at best and not high intensity as reported by httermann and memmert ( 2014 ) . 2015 ) examined the simon task performance in 14 participants while cycling at a low , moderate or very high level of intensity , as defined by the ventilatory threshold . in their study , there was no significant difference in the reaction time ( rt ) across exercise intensities leading the authors to conclude that cognitive control was robust and did not appear to be influenced by the intensity of exercise . there is also debate as to the duration of exercise needed to elicit any change in cognitive performance with studies reporting changes after as little as 6 min of exercise ( mcmorris et al . , 2008 ) to 100 min of exercise ( collardeau et al . there are a number of sports and exercise situations which require high levels of exertion , are aerobically based , but of relatively short duration which facilitate cognitive performance . yet , the impact of exercise intensity during short duration aerobically based exercise has not been widely examined . therefore , given the different conclusions drawn in the literature regarding the effect of exercise intensity on cognition , it is of extreme interest to sport performance practitioners to examine if and when exercise intensity impacts cognitive performance . also of interest is what the actual cut off point ( i.e. exercise intensity ) is before cognitive performance and consequently decision making deteriorates , which may have detrimental consequences for in the field performance . the aim of this study was to examine the effect of short duration , moderate and high intensity exercise on a go / nogo task in habitually trained adults . we hypothesised that cognitive performance during moderate intensity exercise would improve ; however , cognitive performance would be poorer during high intensity exercise compared to rest . following institutional ethics approval and informed consent fifteen habitually trained adults ( 9 females and 6 males , aged 28 5 years ) that trained a minimum of 5 hours per week throughout the last 2 years participated in the study . all participants completed a health history questionnaire to ensure they met all inclusion criteria , i.e. being participants were excluded if they had a musculoskeletal injury or cardiovascular condition which would restrict exercise performance . to measure changes in cognitive performance participants completed a go / nogo task modelled on one developed by pontifex et al . the task used in the present study was a rt paradigm during which subjects performed a binary decision on each stimulus . one of the outcomes required subjects to make a motor response ( go ) , whereas the other required subjects to withhold a response ( no - go ) . the go / nogo task is considered a measure of response inhibition and is generally used to assess the ability to inhibit the prepotent response . specifically , the test required participants to respond quickly and accurately to a circle of 5.5 cm diameter that occurred on 20% of the trials and not to respond to a non - target circle of 3.0 cm diameter that occurred on 80% of the trials . the cognitive task consisted of one unique block of 100 trials performed during the last 2 min of exercise once the target intensity ( high - intensity exercise at 90% of hrr or moderate - intensity at 70% of hrr ) had been reached . stimuli were presented for 300 ms with a 1000 ms interstimulus interval via open source experiment software ( matht et al . , 2012 ) at the centre of a computer monitor located on the treadmill in front of the participant . for each trial , participants were asked to press a trigger button with their dominant hand when the target stimulus was presented . this trigger button process enabled participants to complete the go / nogo task during exercise , thereby addressing key criticism of prior research that studied the effects of exercise on cognitive performance ( lambourne and tomporowski , 2010 ) pre and post exercise . participant s performance on the go / nogo task was calculated and comprised of three measures . two error rates were calculated , one for omission errors relating to instances where the stimulus was presented and the trigger not pressed , and another for decision errors , relating to instances when the non - target stimulus was presented and the trigger was pressed . rts ( ms ) were also calculated for target stimulus trials indicating the time taken to respond when the target stimulus was presented . a resting heart rate ( hrrest ) was also obtained from each participant by getting them to lie down in a prone position for 10 min while wearing a heart rate monitor ( polar rs400 , polar electro oy , kempele , finland ) , in a quiet room void of visual or auditory distractions . a maximum heart rate ( hrmax ) was estimated as 220 minus the participant s age . both the hrrest and hrmax were then recorded and used to calculate 70% and 90% of heart rate reserve ( hrr ) ( karvonen et al . , 1957 ) . the study used a repeated - measures design consisting of three separate sessions performed on different days : rest , moderate intensity and high intensity sessions performed in a counterbalanced order . participants attended the laboratory at the same time of the day in a well - rested and hydrated state with no prior consumption of caffeine or other ergogenic aids that may have influenced cognitive performance . during the initial test session , each participant was allowed 200 attempts at the go / nogo task to familiarise themselves with the test protocol ( pontifex et al . , 2009 ) . an incremental running protocol on a motorised treadmill ( hp cosmos ltd , germany ) was used to induce moderate and high intensity exercise states congruent with previous studies assessing effects of moderate and high intensity exercise on skilled ( lyons et al . , 2008 ) and perceptual ( duncan et al . , 2012 ) performance . whilst in the rest condition , participants stood on the treadmill for a period of approximately 10 min ( the approximate duration for the exercise trials ) before completing the go / nogo task . the workload was then increased by 1.6 km / h every 30/60 s until the participant reached the desired intensity as determined by 70% and 90% of heart rate reserve ( hrr ) ( karvonen et al . , 1957 ) . throughout the test procedures , the borg s ( 1970 ) rating of perceived exertion ( rpe ) scale was also used as an adjunct to monitoring of the heart rate . participants were required to achieve an rpe of 15 - 17 for the moderate intensity exercise condition and 18 - 19 for the high intensity exercise condition . once the desired intensity was reached , as determined by both measures simultaneously ( i.e. % hrr and rpe ) , participants were then required to maintain this intensity for further 2 min . this ensured that participants were truly at the desired steady - state intensity . at this point , participants performed a validated go / nogo task ( pontifex et al . , 2009 ) while still running . each exercise trial lasted approximately 10 min and included approximately 2 min of exercise while completing the go / nogo task . the effects of exercise intensity on error rates ( omission errors and decision errors ) and the rt were analysed using separate 3 ( exercise intensity ) ways repeated measures analysis of variance . where significant differences were found , bonferroni post - hoc pairwise comparisons were used to determine where the differences lay . partial eta squared ( 2 ) was also used as a measure of effect size . the statistical package for social sciences ( spss , version 20 , chicago , il , usa ) was used for all analysis and statistical significance was set , a priori , at p = 0.05 . following institutional ethics approval and informed consent fifteen habitually trained adults ( 9 females and 6 males , aged 28 5 years ) that trained a minimum of 5 hours per week throughout the last 2 years participated in the study . all participants completed a health history questionnaire to ensure they met all inclusion criteria , i.e. being participants were excluded if they had a musculoskeletal injury or cardiovascular condition which would restrict exercise performance . to measure changes in cognitive performance participants completed a go / nogo task modelled on one developed by pontifex et al . the task used in the present study was a rt paradigm during which subjects performed a binary decision on each stimulus . one of the outcomes required subjects to make a motor response ( go ) , whereas the other required subjects to withhold a response ( no - go ) . the go / nogo task is considered a measure of response inhibition and is generally used to assess the ability to inhibit the prepotent response . specifically , the test required participants to respond quickly and accurately to a circle of 5.5 cm diameter that occurred on 20% of the trials and not to respond to a non - target circle of 3.0 cm diameter that occurred on 80% of the trials . the cognitive task consisted of one unique block of 100 trials performed during the last 2 min of exercise once the target intensity ( high - intensity exercise at 90% of hrr or moderate - intensity at 70% of hrr ) had been reached . stimuli were presented for 300 ms with a 1000 ms interstimulus interval via open source experiment software ( matht et al . , 2012 ) at the centre of a computer monitor located on the treadmill in front of the participant . for each trial , participants were asked to press a trigger button with their dominant hand when the target stimulus was presented . this trigger button process enabled participants to complete the go / nogo task during exercise , thereby addressing key criticism of prior research that studied the effects of exercise on cognitive performance ( lambourne and tomporowski , 2010 ) pre and post exercise . participant s performance on the go / nogo task was calculated and comprised of three measures . two error rates were calculated , one for omission errors relating to instances where the stimulus was presented and the trigger not pressed , and another for decision errors , relating to instances when the non - target stimulus was presented and the trigger was pressed . rts ( ms ) were also calculated for target stimulus trials indicating the time taken to respond when the target stimulus was presented . a resting heart rate ( hrrest ) was also obtained from each participant by getting them to lie down in a prone position for 10 min while wearing a heart rate monitor ( polar rs400 , polar electro oy , kempele , finland ) , in a quiet room void of visual or auditory distractions . a maximum heart rate ( hrmax ) was estimated as 220 minus the participant s age . both the hrrest and hrmax were then recorded and used to calculate 70% and 90% of heart rate reserve ( hrr ) ( karvonen et al . , 1957 ) . the study used a repeated - measures design consisting of three separate sessions performed on different days : rest , moderate intensity and high intensity sessions performed in a counterbalanced order . participants attended the laboratory at the same time of the day in a well - rested and hydrated state with no prior consumption of caffeine or other ergogenic aids that may have influenced cognitive performance . during the initial test session , each participant was allowed 200 attempts at the go / nogo task to familiarise themselves with the test protocol ( pontifex et al . , 2009 ) . an incremental running protocol on a motorised treadmill ( hp cosmos ltd , germany ) was used to induce moderate and high intensity exercise states congruent with previous studies assessing effects of moderate and high intensity exercise on skilled ( lyons et al . , 2008 ) and perceptual ( duncan et al . , 2012 ) performance . whilst in the rest condition , participants stood on the treadmill for a period of approximately 10 min ( the approximate duration for the exercise trials ) before completing the go / nogo task . the workload was then increased by 1.6 km / h every 30/60 s until the participant reached the desired intensity as determined by 70% and 90% of heart rate reserve ( hrr ) ( karvonen et al . , 1957 ) . throughout the test procedures , the borg s ( 1970 ) rating of perceived exertion ( rpe ) scale was also used as an adjunct to monitoring of the heart rate . participants were required to achieve an rpe of 15 - 17 for the moderate intensity exercise condition and 18 - 19 for the high intensity exercise condition . once the desired intensity was reached , as determined by both measures simultaneously ( i.e. % hrr and rpe ) , participants were then required to maintain this intensity for further 2 min . this ensured that participants were truly at the desired steady - state intensity . at this point , participants performed a validated go / nogo task ( pontifex et al . , 2009 ) while still running . each exercise trial lasted approximately 10 min and included approximately 2 min of exercise while completing the go / nogo task . the effects of exercise intensity on error rates ( omission errors and decision errors ) and the rt were analysed using separate 3 ( exercise intensity ) ways repeated measures analysis of variance . where significant differences were found , bonferroni post - hoc pairwise comparisons were used to determine where the differences lay . partial eta squared ( 2 ) was also used as a measure of effect size . the statistical package for social sciences ( spss , version 20 , chicago , il , usa ) was used for all analysis and statistical significance was set , a priori , at p = 0.05 . results indicated significant exercise intensity main effects for the rt ( f2 , 28 = 6.169 , p = 0.01 , partial 2 = 0.320 , figure 1 ) . bonferroni post - hoc pairwise comparisons indicated significantly slower rts during high intensity exercise compared to rest ( p = 0.023 ) and moderate intensity exercise ( p = 0.039 ) . the omission error rate ( f2 , 28 = 4.108 , p = 0.027 , partial 2 = 0.255 ) and decision error rate ( f2 , 28 = 9.213 , p = 0.011 , partial 2 = 0.397 ) were also significantly different across exercise intensities . post - hoc analysis indicated a significantly higher omission error rate at high intensity compared to moderate ( p = 0.038 ) intensity exercise and rest ( p = 0.043 ) . this pattern was replicated for the decision error rate with higher decision errors being made at high intensity exercise compared to moderate intensity ( p = 0.008 ) and rest ( p = 0.002 ) . mean se of go / nogo task rts ( ms ) to target stimuli during rest , moderate and high intensity treadmill running ( * p = 0.023 , * * p = 0.039 ) mean se of go / nogo task omission error and decision error rates ( % ) to target stimuli during rest , moderate and high intensity treadmill running the aim of the present study was to examine the effect of exercise intensity on a go / nogo task performance . ( 2012 ) who investigated the relationship between running speeds of 4.8 , 8.0 and 12.8 km / h , exercise intensities of 70% and 90% of hrr and cognitive performance , with the largest decrements in cognitive performance taking place at 12.8 km / h and exercise intensity of 90% of hrr , but do not align with other studies suggesting that moderate intensity exercise results in enhanced cognitive performance ( chmura et al . , 1994 ; mcmorris and graydon , 2000 ; mcmorris et al . , 2011 ) . cognitive performance is significantly impaired during high intensity exercise ( 90% of hrr ) which extends the findings proposed by httermann and memmert ( 2014 ) who failed to reach an exercise intensity at which cognitive performance was negatively affected in trained athletes . the results of the current study are also contrary to those recently published by davranche et al . ( 2015 ) which reported no effect of exercise intensity on the simon task performance during a 20 min cycling task . moreover , davranche et al . ( 2015 ) highlighted in their study that there was no sign of worsening rts during very high intensity exercise . from a sporting point of view , it is extremely important to determine at what point cognitive performance is negatively affected by exercise intensity and the resulting fatigue . for example , in soccer it is well known that a large number of goals are scored in the final minutes of a game when the relationship between fatigue and decision making of some players may be at their poorest . consequently , further investigation is required so that a clearer theoretical explanation can be proposed which will help clarify the effects of exercise intensity on cognitive performance . a number of authors who have proposed a theoretical explanation for effect of exercise intensity on cognitive performance have suggested that acute aerobic exercise is an arousing stressor ( audiffren , 2009 ) and as such the theoretical explanations have been anchored in unidimensional theories of arousal including the inverted - u theory ( yerkes and dodson , 1908 ) . however , if arousal is assumed to be a mechanism by which performance changes , then some form of explanation is also required for the role of cognition in this process ( hardy and parfitt , 1991 ) . it may be that due to the multidimensional construct of arousal , which has a cognitive and physiological component , the catastrophe model ( fazey and hardy , 1988 ) may provide a more accurate account of the relationship between cognitive performance and exercise intensity . specifically , the catastrophe model predicts that when physiological arousal and cognitive anxiety are low , performance will follow an inverted - u , which has been reported before ( chmura et al . , 1994 ; mcmorris and graydon , 2000 ; mcmorris et al . , 2011 however , when physiological arousal and cognitive anxiety are at their highest , the effect on performance will be at its worst which is what was found in the present study at an exercise intensity of 90% . although cognitive anxiety was not measured in the present study , it can be assumed that due to extremely demanding nature of the physiological component ( i.e. 90% ) of the study that cognitive anxiety would be at its highest , which goes some way in supporting the predictions of the catastrophe model ( fazey and hardy , 1988 ) . such intensity of exercise has also been suggested to be anxiety provoking in the study by davranche et al . ( 2015 ) . in this context , higher intensity exercise coupled with performance of cognitive tasks may result in increased demands of the concurrent activities leading to greater demand on attentional resources and potentially poorer performance . ( 2007 ) found that there was a prioritization for postural control over the cognitive stimulus when the highest threat was to postural stability . 2007 ) suggested that it was not until the appropriate postural responses had been initiated ( or inhibited ) that the cognitive stimulus could be completely attended to . in the context of the present study , an exercise intensity of 90% the results of the present study would broadly support the assertion that allocation of attentional resources is inhibited during high intensity exercise resulting in poorer cognitive task performance compared to lower exercise intensities . furthermore , such an argument may explain the discrepancy between the results presented in the current study and those of davranche et al . , the use of a cycle based exercise modality reduced the postural prioritisation effect , unlike the current study where postural prioritisation during treadmill running may have been more of a factor . indeed in their meta - regression analysis , lambourne and tomporowski ( 2010 ) had previously reported that cycle and treadmill based exercise produced different results in respect to effects of exercise intensity on cognitive performance . in the present study , it is possible that when running at high intensity a speed accuracy trade - off was evidenced as , in order to continue to run and remain upright , rts worsened and additional errors were made .. it is also important to note that during moderate and high intensity exercise conditions , the cognitive workload required to perform at such velocity on a treadmill is very high . thus , when comparing the resting condition with the exercise conditions , there is an effect of exercise and a strong dual task effect . this may be one reason why there was no observation of facilitation in the moderate intensity condition and impaired performance in the high intensity condition . the use of a 10 min rest period as a control condition in the present study was undertaken to provide a true rest period of the same duration as the exercise bouts and to avoid comparing to rest pre - exercise where there may have been anticipatory responses as a result of the upcoming tasks . for future studies , a more relevant control condition could comprise of very low exercise intensity rather than simply standing on a treadmill . until now the focus of the argument has mainly revolved around the effects of changes in exercise intensity on cognitive performance with very little discussion on the influence of the cognitive test on performance outcomes . the importance of choosing an appropriate cognitive test can not be underestimated , for example , memmert et al . ( 2009 ) who investigated the relationship between visual attention and expertise in sport using a functional field of a view task , a multiple - object tracking task , and an attentional blindness task found that team sports experts showed no better performance on the basic attention tasks than athletes from non - team sports or novice athletes which is unlike the httermann and memmert ( 2014 ) and the present study where significant differences in cognitive performancewere reported 2009 ) failed to include any form of a physiological component in their study and although the authors suggested that any attentional focus task that reveal group differences could potentially be used to design training programs to improve sport - specific attention capacity , we propose that this proposal should be viewed with caution unless some form of exercise intensity is included . we also acknowledge that the go / nogo test of cognitive performance employed in the present study was simple in nature and unlike the protocol used in the httermann and memmert s ( 2014 ) study that used a more cognitively demanding attentional breadth cognitive test , which may be the reason for the disparity of results between the present study and that of hutterman and memmert ( 2014 ) . a more complex version of the same go / nogo task used in the present study is available ( pontifex et al . , 2009 ) which includes an additional square distracter stimulus which may provide a more detailed account of cognitive performance . however , a cautionary note is that in the present study it was difficult to utilise a more complex cognitive performance test while running at 90% of hrr due to the trade - off between the time requirement to complete the test and the physical capacity of individuals to remain running at this intensity . therefore , future research will seriously need to consider alternative and effective ways that can both ensure the safety of the participant while exercising at high intensities whilst at the same time completing more difficult and demanding cognitive tests . a further limitation in the present study is that the thresholds for moderate and high intensity exercise were calculated using the karvonen formula ( karvonen et al . , 1957 ) . this was employed in order to account for individual variation in a resting heart rate as a consequence of different fitness levels in participants . the use of the karvonen formula in the present study was also chosen because it is recommended as a means to set a target heart rate by the american college of sports medicine ( 2006 ) and had been cited in prior studies reporting the effect of moderate and high intensity exercise on cognitive performance ( borg , 1970 ; duncan et al . , 2012 ; lyons et al . , 2008 however , future studies may be more effective by either using an alternative equation to estimate the maximum heart rate or by establishing exercise intensity as a percentage of vo2max . in conclusion , the present study suggests that high intensity exercise results in poorer cognitive performance in habitually active adults compared to rest or moderate intensity exercise . to accurately understand the exercise intensity and cognitive performance relationship in athletes , research must adopt a multidimensional approach that includes both a high exercise intensity condition of at least 90% of either vo2max or hrr and an equally demanding cognitive task that is transferable to actual sport performance .

abstractthis study examined the effect of short duration , moderate and high - intensity exercise on a go / nogo task . fifteen , habitually active ( 9 females and 6 males aged 28 5 years ) agreed to participate in the study and cognitive performance was measured in three sessions lasting 10 min each , performed at three different exercise intensities : rest , moderate and high . results indicated significant exercise intensity main effects for reaction time ( rt ) ( p = 0.01 ) , the omission error rate ( p = 0.027 ) and the decision error rate ( p = 0.011 ) , with significantly longer rts during high intensity exercise compared to moderate intensity exercise ( p = 0.039 ) and rest ( p = 0.023 ) . mean se of rt ( ms ) was 395.8 9.1 , 396.3 9.1 and 433.5 16.1 for rest , moderate and high intensity exercise , respectively . this pattern was replicated for the error rate with a significantly higher omission error and decision error rate during high intensity exercise compared to moderate intensity exercise ( p = 0.003 ) and rest ( p = 0.001 ) . mean se of omission errors ( % ) was 0.88 0.23 , 0.8 0.23 and 1.8 0.46% for rest , moderate and high intensity exercise , respectively . likewise , mean se of decision errors ( % ) was 0.73 0.24 , 0.73 0.21 and 1.8 0.31 for rest , moderate and high intensity exercise , respectively . the present study s results suggest that 10 min workout at high intensity impairs rt performances in habitually active adults compared to rest or moderate intensity exercise .

Introduction Material and Methods Participants Measures Procedure Analysis Results Discussion

PMC5154713

regular physical activity is an important for the prevention and treatment of overweight and obesity . although physical activity is not the main means for reducing body fat , it improves cardio - respiratory fitness and lowers the risk of heart disease , among others , if performed regularly and safely ( 9 , 16 ) . despite the knowledge of the benefits of exercise , overweight and obese individuals tend to be sedentary ( 1 , 3 ) . regarding their low level of physical activity , it has been observed that the way the individual perceives physical exercise tends to influence their participation ( 36 ) . in this sense , the development of strategies that encourage exercise to be perceived more positively may be interesting for this population . music is an important way to reduce monotony during exercise ( 17 , 21 , 30 ) . its use can produce psychological and physiological effects , influencing the rating of perceived exertion ( rpe ) and the attentional focus during exercise ( 21 ) . the rpe can be defined as the subjective intensity of effort , strain , discomfort , and/or fatigue that is experienced during physical exercise ( 32 ) . in the context of endurance exercise these terms are dichotomous , while associative attentional focus has been defined as attention in physical sensations or other task - related process , dissociative attentional focus has been defined as attention in sensations or task - not related with the physical work ( 7 , 9 ) . despite knowledge of the effect of music on psychological and psychophysical responses , the use of music seems to influence the rpe when compared to no - music conditions . in a study conducted by potteiger , schroeder , and goff ( 29 ) , the use of music produced a reduction in all conditions when compared with the no - music condition . moreover , almeida et al . ( 3 ) found that the rpe is higher with fast tempo music compared with the no - music and medium - tempo music conditions . when the exercise is performed at low or moderate intensity , external factors , like music , can be process in parallel with physiological cues , attenuating this response on the information process . however , at higher intensities , physiological cues seem to dominate the processing of information ; while at moderate intensity , external routes , such as the use of music , can be processed in parallel ( 31 , 33 ) . accordingly , a motivational factor of music tempo appears to influence rpe . regarding attentional focus , previous studies have observed that elite runners use an associative attentional focus during a race whereas recreational runners have dissociative focus during the effort ( 27 ) . the associative attentional focus seems to be more productive in highly conditioned individuals because they are able to meet the physiological demands objectively ( 8 , 11 ) . however , sedentary subjects with lower fitness levels can benefit from a dissociative attentional focus , reducing bodily sensations and positively interpreting the effort . in this manner , strategies to modulate the rpe and make the attentional focus more dissociative could benefit lower - conditioned people . this study observed the effect of music on the rpe and the attentional focus in self - selected paced walking performed by overweight and obese women . a sample of 15 women aged 35 to 50 participated in the study ( mean sd , age 42.9 4.9 years , height 1.56 0.07 m , body mass 79.7 11.9 kg , body mass index 32.7 5.1 kg / m , peak oxygen uptake 24.7 4.7 ml.kg.min . ) . inclusion criteria were as follows : ( a ) engages in less than 30 minutes of exercise on most days of the week ( 1 ) ; ( b ) has a body mass index ( bmi ) of between 25 and 39 kg / m ; ( c ) does not make use of pharmacological resources that could change the outcome of the study ; ( d ) does not have cardiovascular disease ; ( e ) answers negative to all questions in the physical activity readiness questionnaire ( par - q ) . this study was approved by the research ethics committee of the federal university of paran , and all participants signed an informed - consent form before starting the research . the medium tempo was between 115 and 120 beats per minute ( bpm ) while the fast tempo was between 140 and 145 bpm ( 21 ) . the participants were asked to prepare a list of their three favorite artists , in hierarchical order , to exercise context . upon completion of the list , three medium - tempo and three fast - tempo songs from each artist were used for the experimental sessions ( 22 ) . body weight ( kg stadiometer sanny , so paulo , brazil ) and height ( cm , toledo , sao paulo , brazil ) were measured according to the technique described by lohman , roche , and martorell ( 26 ) . to calculate the body mass index ( bmi ) the following formula was used : kg / m . this instrument essentially comprises a category rating scale of 15 points , with scores ranging from 6 ( minimal effort ) to 20 ( maximum effort ) . the rpe has been analyzed and related to several physiological variables such as heart rate , oxygen uptake , blood lactate , metabolic acidosis , among others markers ( 5 , 24 ) . the attentional focus was measured by the attentional focus questionnaire - afq ( 8) . this questionnaire evaluates the focus of attention asses how much exercisers think about some activities along three domains ( association , dissociation and distress ) , comprising 30 questions , with answers ranging from 1 ( not ever ) to 7 ( all the time ) , divided into dissociative attention , associative attention , and distress . for the study , we considered only the associative and dissociative attentional domains . some studies has been discussed the psychometric proprieties and utilized afq to evaluate the attentional focus from exercise ( 25 , 35 ) . the participants performed an incremental maximal test ( tmax ) using the bruce protocol ( 10 ) . the test started at a speed of 2.7 kilometer per hours ( km / hr ) and a 10% slope . every three minutes there was a load increment on both the inclination and the speed to increase participants volitional fatigue . the tmax was performed on a reebok fitness treadmill ( x fit-7 model , london , uk ) . the heart rate ( hr , beats per min ) was measured by a polar monitoring system ( polar electro oy , kempele , finland ) . during the test , oxygen uptake ( vo2 ) was measured using the k-4 system ( cosmed , k4b2 , rome , italy ) in the process peak oxygen uptake ( vo2peak ) was considered the greatest vo2 achieved during the last 30 seconds of the test . maximum heart rate ( hrmax ) was defined as the highest value obtained for the hr during the tmax . this study comprised four meetings : ( a ) familiarization , anthropometric measures and maximal exercise test ( laboratory environment ) ; ( b ) three walking sessions in the external environment , with medium - tempo music , fast - tempo music , and no - music conditions . participants were requested not to perform physical exercises 24 hours before each workout and not to have energy drinks , coffee , or caffeinated products in a period of three hours before each session ( 2 ) . at first meeting , the participants were asked to fill all information and assign the consentient to participate on the research . rpe scale and afq were shown to participants and they were instructed how to use them . a period of 72 hours after performing the incremental maximal test was set for the participants to performed the first of the three experimental sessions . each session had one of three conditions : walking with use of fast - tempo music ( ft ) ; walking with medium - tempo music ( mt ) ; and no - music control ( nm ) . the experimental sessions were performed on external environment , on a standard outdoor 400-m track . each session comprised a three - minute of warm - up , 30 minutes of walking at a self - selected intensity and five minutes of cool down . at warm - up , the participants were instructed to walking at an intensity that would prepare them to exercise . at begin of each experimental session , were given the following instruction : you will be allowed to select an intensity you prefer to perform on overground . this should be an intensity that you would choose for a 30-min workout if you were participating in a fitness program . the intensity should be high enough that you would get a good workout , but not so high that you would not prefer to exercise at that intensity daily or at least every other day . after the first 5 min , they were allowed to adjust their walking speed only every 5 min of the 20-min trial ( i.e. , minutes 5:00 , 10:00 , 15:00 , 20:00 and 25:00 ) . for cool - down , the participants were instructed to walking at a lower intensity that they performed during the 30 minute of exercise , at a self - selected pace that made them decelerate . experimental sessions were conducted individually to avoid effect of group dynamics or social interaction on the psychophysiological responses to exercise ( 13 ) . the temperature ( degree celsius , c ) was 22.53 3.24 ; 22.20 3.32 and 23.06 2.52 , for nm , mt and ft , respectively . the percent relative humidity of air ( % ) was 51.26 0.96 ; 51.53 0.83 and 51.26 0.96 for nm , mt and ft , respectively . the order of the sessions was randomized , and participants were not provided with information about the music tempo in sessions spent with the use of music . in the sessions with music , when the participants walking without music , they received the headphone , with this not playing sound . during the experimental sessions , the physiological responses were determined breath by breath and converted to percentages of vo2peak ( % vo2peak ) and hrmax ( % hrmax ) . the rpe was measured at 5 , 10 , 15 , 20 , 25 , and 30 minutes of exercise . six points were considered : five - minute averages were used for comparison between conditions ( 01:0005:00 , 06:0010:00 , 11:0015:00 , 16:0020:00 , 21:0025:00 and 26:0030:00 ) . to rpe analysis , were utilized the six time points that they were collected and calculated average of session . to afq analysis , the sum of each variable ( dissociation and association ) it was utilized to compare the conditions . data are presented as mean standard error ( se ) . for all analysis ( physiological , perceptual and attentional focus ) , the repeated measures anova compared the three conditions : medium - tempo music ; fast - tempo music ; no - music control . in the presence of violations in the sphericity assumptions , when significance was found , bonferroni s post - hoc was utilized to find where was difference . a sample of 15 women aged 35 to 50 participated in the study ( mean sd , age 42.9 4.9 years , height 1.56 0.07 m , body mass 79.7 11.9 kg , body mass index 32.7 5.1 kg / m , peak oxygen uptake 24.7 4.7 ml.kg.min . ) . inclusion criteria were as follows : ( a ) engages in less than 30 minutes of exercise on most days of the week ( 1 ) ; ( b ) has a body mass index ( bmi ) of between 25 and 39 kg / m ; ( c ) does not make use of pharmacological resources that could change the outcome of the study ; ( d ) does not have cardiovascular disease ; ( e ) answers negative to all questions in the physical activity readiness questionnaire ( par - q ) . this study was approved by the research ethics committee of the federal university of paran , and all participants signed an informed - consent form before starting the research . the medium tempo was between 115 and 120 beats per minute ( bpm ) while the fast tempo was between 140 and 145 bpm ( 21 ) . the participants were asked to prepare a list of their three favorite artists , in hierarchical order , to exercise context . upon completion of the list , three medium - tempo and three fast - tempo songs from each artist were used for the experimental sessions ( 22 ) . body weight ( kg stadiometer sanny , so paulo , brazil ) and height ( cm , toledo , sao paulo , brazil ) were measured according to the technique described by lohman , roche , and martorell ( 26 ) . to calculate the body mass index ( bmi ) this instrument essentially comprises a category rating scale of 15 points , with scores ranging from 6 ( minimal effort ) to 20 ( maximum effort ) . the rpe has been analyzed and related to several physiological variables such as heart rate , oxygen uptake , blood lactate , metabolic acidosis , among others markers ( 5 , 24 ) . the attentional focus was measured by the attentional focus questionnaire - afq ( 8) . this questionnaire evaluates the focus of attention asses how much exercisers think about some activities along three domains ( association , dissociation and distress ) , comprising 30 questions , with answers ranging from 1 ( not ever ) to 7 ( all the time ) , divided into dissociative attention , associative attention , and distress . for the study , we considered only the associative and dissociative attentional domains . some studies has been discussed the psychometric proprieties and utilized afq to evaluate the attentional focus from exercise ( 25 , 35 ) . the participants performed an incremental maximal test ( tmax ) using the bruce protocol ( 10 ) . the test started at a speed of 2.7 kilometer per hours ( km / hr ) and a 10% slope . every three minutes there was a load increment on both the inclination and the speed to increase participants volitional fatigue . the tmax was performed on a reebok fitness treadmill ( x fit-7 model , london , uk ) . the heart rate ( hr , beats per min ) was measured by a polar monitoring system ( polar electro oy , kempele , finland ) . during the test , oxygen uptake ( vo2 ) was measured using the k-4 system ( cosmed , k4b2 , rome , italy ) in the process peak oxygen uptake ( vo2peak ) was considered the greatest vo2 achieved during the last 30 seconds of the test . maximum heart rate ( hrmax ) was defined as the highest value obtained for the hr during the tmax . this study comprised four meetings : ( a ) familiarization , anthropometric measures and maximal exercise test ( laboratory environment ) ; ( b ) three walking sessions in the external environment , with medium - tempo music , fast - tempo music , and no - music conditions . participants were requested not to perform physical exercises 24 hours before each workout and not to have energy drinks , coffee , or caffeinated products in a period of three hours before each session ( 2 ) . at first meeting , the participants were asked to fill all information and assign the consentient to participate on the research . rpe scale and afq were shown to participants and they were instructed how to use them . a period of 72 hours after performing the incremental maximal test was set for the participants to performed the first of the three experimental sessions . each session had one of three conditions : walking with use of fast - tempo music ( ft ) ; walking with medium - tempo music ( mt ) ; and no - music control ( nm ) . the experimental sessions were performed on external environment , on a standard outdoor 400-m track . each session comprised a three - minute of warm - up , 30 minutes of walking at a self - selected intensity and five minutes of cool down . at warm - up , the participants were instructed to walking at an intensity that would prepare them to exercise . at begin of each experimental session , were given the following instruction : you will be allowed to select an intensity you prefer to perform on overground . this should be an intensity that you would choose for a 30-min workout if you were participating in a fitness program . the intensity should be high enough that you would get a good workout , but not so high that you would not prefer to exercise at that intensity daily or at least every other day . after the first 5 min , they were allowed to adjust their walking speed only every 5 min of the 20-min trial ( i.e. , minutes 5:00 , 10:00 , 15:00 , 20:00 and 25:00 ) . for cool - down , the participants were instructed to walking at a lower intensity that they performed during the 30 minute of exercise , at a self - selected pace that made them decelerate . experimental sessions were conducted individually to avoid effect of group dynamics or social interaction on the psychophysiological responses to exercise ( 13 ) . the temperature ( degree celsius , c ) was 22.53 3.24 ; 22.20 3.32 and 23.06 2.52 , for nm , mt and ft , respectively . the percent relative humidity of air ( % ) was 51.26 0.96 ; 51.53 0.83 and 51.26 0.96 for nm , mt and ft , respectively . the order of the sessions was randomized , and participants were not provided with information about the music tempo in sessions spent with the use of music . in the sessions with music , when the participants walking without music , they received the headphone , with this not playing sound . during the experimental sessions , the physiological responses were determined breath by breath and converted to percentages of vo2peak ( % vo2peak ) and hrmax ( % hrmax ) . the rpe was measured at 5 , 10 , 15 , 20 , 25 , and 30 minutes of exercise . to physiological analysis , six points were considered : five - minute averages were used for comparison between conditions ( 01:0005:00 , 06:0010:00 , 11:0015:00 , 16:0020:00 , 21:0025:00 and 26:0030:00 ) . to rpe analysis , were utilized the six time points that they were collected and calculated average of session . to afq analysis , the sum of each variable ( dissociation and association ) it was utilized to compare the conditions . data are presented as mean standard error ( se ) . for all analysis ( physiological , perceptual and attentional focus ) , the repeated measures anova compared the three conditions : medium - tempo music ; fast - tempo music ; no - music control . in the presence of violations in the sphericity assumptions , when significance was found , bonferroni s post - hoc was utilized to find where was difference . with regarding physiological responses , there were no differences between conditions for % vo2peak ( f2:30= 0.329 ; p= 0.723 ; p= 0.29 ) or % hrmax ( f2:30= 0.904 ; p= 0.382 ; p= 0.076 ) . nm , mt and ft were performed under 77.45 2.89 ; 75.14 2.91 and 75.98 2.86 % hrmax , respectively . with regarding % vo2peak , nm , mt and ft performed at 59.04 3.48 , 56.81 3.81 and 58.00 3.90 , respectively . anova found condition effect in perceptual responses ( f2:28= 4.380 ; p= 0.02 ; p= 0.238 ) . the post - hoc bonferroni found that rpe it was lower at mt than at nm ( 10.5 0.5 vs. 12.05 0.6 , respectively ) . there were no differences between ft and mt ( 10.9 0.4 vs. 10.5 0.5 , respectively ) or between ft and nm ( 10.9 0.4 vs. 12.05 0.6 , respectively ) . attentional focus is present in figure 2 . regarding the dissociative focus , there was effect of condition ( f2:30= 5.975 ; p= 0.008 ; p= 0.352 ) . the post - hoc bonferroni found that nm was lower than mt and ft ( 39.0 4.1 ; 48.4 4.1 and 47.9 4.5 , respectively ) . the anova found no effect of the condition on associative focus ( f2:30= 0.013 ; p= 0.987 ; p= 0.001 ) . this study observed the effect of music on the rpe and attentional focus at a self - selected paced exercise sessions performed by overweight and obese women . the results of this study were as follows : rpe was greater in the no - music control compared with that in medium - tempo music ; the dissociative attentional focus was higher in conditions with medium and fast tempo music compared with the no - music control ; no differences were observed among the three conditions in associative attentional focus . reducing the rpe through the use of music has been found in previous studies ( 29 ; 34 ) . however , this effect depends on the intensity at which the exercise is performed . at high intensities , the physiological feedback from exercise seems to dominate the processing of information for the modulation of the rpe . on the other hand , at low and moderate intensities , external factors such as the use of music can be processed in parallel in the process of the rpe ( 8 , 11 ) . the intensity of walking sessions were self - selected paced , that were performed at moderate intensity ( 1 ) . in this sense , using music in low- or moderate - intensity exercise , such as walking , can be interesting for the participants to experience exercise in a more positive way . the fact that the rpe shows no differences between ft and nm may be due to the stimulus exposure time . in fact , the literature shows that the tempo of music can influence the rpe of the subject during physical exercise ( 3 ) . ( 21 ) suggest that continuous exposure to fast tempo music may result in reduced motivation , leading to loss of interest in the activity . the relevance of the stimulus is the causative factor in changing the attentional focus , that can be explained by social - cognitive perspective of perceived exertion and exertion tolerance ( 33 ) . with low or moderate intensity , the focus on stimuli unrelated to the exercise predominates in the formation of cognitive response because the exercise has been undertaken lightly . on the other hand , at higher intensities the brain shifts attention to internal cues information due to changes caused by the increased exercise intensity . our results can confirm this theory , where the session s intensity were at moderate intensity ( 1 ) . dissociative attentional focus has been received attention because your association with factor linked at adherence to exercise ( like affective response , enjoyment , and others ) . is well established that if the exercise is not perceived as enjoyable , tolerable or comfortable , probably individuals will not repeat the activity and will drop out the training ( 15 ) . the dissociative strategies can be a solution to how the exercise stimulus is experienced ( 25 ) . dissociative techniques have been suggested to diverting attention away from uncomfortable or displeasure stimulus during exercise . moreover , dissociative focus has been positively associated with revitalization , positive engagement , and reductions in physical exhaustion during exercise ( 23 ) . for example pennebaker and lightner ( 28 ) found that external focus was better enjoyment and satisfaction compared to internal focus during aerobic exercise . another question , is that manipulating attentional focus during exercise , like music use , for example , can influence feeling of pleasure and enjoyment during exercise session , even when at intensities slightly above from ventilatory threshold ( 20 ) . dissociative attentional focus has been suggested improve both immediate and long - term exercise program adherence ( 14 ) . however , few studies has been investigated the relationship between dissociative attentional focus and adherence or maintenance . annesi ( 4 ) found that dissociative attentional focus during exercise had significantly lower drop out , as well as trend toward to higher attendance than control condition . nonetheless , the causal effects between dissociative attentional focus and adherence need of experimental designs to be explained . the present sample was comprised of overweight and obese women adults and thus the findings can not be readily generalized to the other population without replicate the same design . the benefit of exercising outdoors versus indoors has been established in the literature ( 12 ) . the present participants walking at an external environment in relative isolation , and it is noteworthy that inmost real life exercise environments . the present findings show that at moderate exercise intensity , external factors can modulate psychological responses . the use of music while walking at a self - selected paced intensity can improve dissociative attentional focus during exercise . moreover , medium tempo music can lower perceived exertion in overweight and obese individuals . our results have practical implication for people that could benefit from strategies that cause a lower interpretation from internal cues during exercise , as overweight and obese women . for future studies , we suggest designs that use dissociative strategies on specific people , as obese man , elderly and sedentary .

this study investigated the influence of music on the rating of perceived exertion ( rpe ) and attentional focus during walking at a self - selected pace . fifteen overweight and obese women volunteered to participate in the study . they underwent four sessions : the first for incremental maximal test and anthropometric measurement followed by three experimental sessions . after the first session , they were exposed to three 30-minute walking sessions at a self - selected pace in a counterbalanced order : fast - tempo music ( ft ) , medium - tempo music ( mt ) and no - music control ( nm ) . borg s rpe scale and an attentional focus questionnaire were used to measure the perceptual response and attentional focus , respectively . results showed that the rpe was higher in the no - music control than in the medium - tempo music ( 12.05 0.6 vs. 10.5 0.5 ) . furthermore , dissociative attentional focus was greater for both conditions with music in comparison with the no - music control ( nm= 39.0 4.1 ; mt= 48.4 4.1 and ft= 47.9 4.5 ) . the results indicated that the use of music during walking can modulate attentional focus , increasing dissociative thought , and medium - tempo music can reduce the rpe .

INTRODUCTION METHODS Participants Protocol Statistical Analysis RESULTS DISCUSSION

PMC4590904

iliotibial band syndrome is a common knee injury caused by inflammation of the distal portion of the iliotibial band ( itb ) , which results in lateral knee pain . the distal iliotibial band slides over the lateral femoral epicondyle , and during repetitive flexion and extension activities of the knee excessive friction and potential irritation results in pain . potential risk factors for the development of iliotibial band syndrome include preexisting iliotibial band tightness , high weekly mileage , time spent walking or running on a track , interval training , and muscular weakness of knee extensors , flexors , and hip abductors [ 1 , 2 ] . populations who expose their knees to a greater amount of flexion and extension activities , such as athletes , particularly long distance athletes , put themselves at a higher risk for iliotibial band syndrome . due to the pathophysiology of it band syndrome , runners have been a group often looked at for prevalence and management of this syndrome . itb syndrome has been documented to have as high as a 22.2% incidence of all lower extremity injuries in runners . despite a clear pathophysiology , few studies have shown any direct relationship between biomechanical factors and the development of iliotibial bad syndrome [ 1 , 2 , 46 ] . athletes with itb syndrome typically complain of a sharp or burning pain roughly 2 cm superior to the lateral joint line . the pain may radiate proximally or distally , and in less severe cases , the pain may quickly subside upon cessation of activities . often pain will occur as activities proceed . it is not uncommon that the athlete will experience popping on the lateral aspect of the knee with activities . itb syndrome is a clinical diagnosis and most often additional diagnostic studies are not necessary . it should be suspected in overuse and nontraumatic cases of knee pain where rest has not been helpful . ober 's test is one of numerous physical exam tests often used to assess the tightness of the itb . if the leg can be passively stretched to a position horizontal but not completely adducted to a table , this constitutes minimal tightness . if the leg can be passively adducted to horizontal at best , this constitutes moderate tightness , and if it can not be passively adducted to horizontal , this is maximal tightness . popping of the itb over the lateral femoral condyle can also occur in this position as the knee is brought through range of motion . history , however , is much more important than physical exam in diagnosis and short - term resolution of symptoms following corticosteroid injection and be both diagnostic and therapeutic . mri may be of use if there is doubt about the diagnosis as well as to exclude an intra - articular problem such as a lateral meniscal tear ; however , isolated itbfs often does not lead to mri abnormalities and can be misdiagnosed if a minor but different lesion is present . two studies revealed that in patients with iliotibial band syndrome , mri studies have shown that the distal portion of the itb may thicken , and a bursa deep to the iliotibial band over the lateral epicondyle becomes inflamed and filled with fluid [ 8 , 9 ] . when the athletic population was isolated , normal , or cystic , poorly defined signal intensities at the distal portion of the itb predominated . only in chronic cases was a thickening of the distal itb at the level of the lateral femoral epicondyle seen . while the majority of patients respond to a nonsurgical , conservative approach , this does not occur for all , and escalation of treatment is necessary . this is especially true in athletes that present with refractory cases , and at this time surgical intervention can be used . unfortunately , refractory cases can occur quite often , and no treatment has been shown to work best . the purpose of this review is to outline both the conservative and surgical options for treatment of iliotibial band syndrome in athletes . in order to find the most current treatment options for itb syndrome in athletes , a literature search was conducted in the pubmed database . criteria for inclusion in this review were papers that primarily ( but not exclusively ) focused on the athletic population , achieved a level iii or greater level of evidence , addressed therapeutic options for itb syndrome ( conservative or surgical ) , and were written in english . an initial search of iliotibial band syndrome yielded 176 results . after limiting the results to those articles that discussed treatment options , and focused on the athletic population many of these treatment modalities have been geared toward the runner population , and certain guidelines to return to sport ( running ) have been suggested . table 1 illustrates four studies that outlined conservative treatment modalities . in a randomized controlled trial ( rct ) , schwellnus et al . investigated the effect of initial treatment ( day 07 : rest , ice application , and medication ) in 43 patients with unilateral itbs . all subjects received physical therapy consisting of ultrasound , deep transverse friction massages on days 3 , 5 , and 7 and daily stretching of the it band . medication was delivered over the 7 days in a double - blind , placebo - controlled fashion with group 1 taking a placebo anti - inflammatory , group 2 an anti - inflammatory ( voltaren ) , and group 3 an anti - inflammatory / analgesic ( myprodol ) . compared with the other groups , group 3 had less pain during running from day 3 onward , and their running time / distance on the treadmill significantly increased from day 0 to 7 . in another randomized controlled trial , 18 runners with acuteonset itb syndrome ( < 14 days ' duration ) were randomly assigned into two groups : group 1 received a corticosteroid injection and group 2 received a placebo injection . subjects were instructed not to run for 14 days following the injection and to apply ice to the area for 30 minutes every 12 hours . running pain was significantly decreased in the group that received the corticosteroid injection . in a case series of 196 running injuries , pinshaw et al . found itbs to the third most common injury ( 12% ) behind peripatellar pain syndrome ( 22% ) and posterior tibial stress syndrome ( 18% ) . in those with these steps included running shoes : change to softer shoes , use of in - shoe support and shoe alterations , and/or removal of the outside heel flare of the shoe for the injured side . leg - length discrepancies : adapt shoe of the shorter leg by adding material to the mid - sole to ensure 100% correction at the heel , 50% correction in the mid - sole , and 25% correction at the ball of the foot . training methods : if appropriate , reduce training distance and decrease running speed and amount of hill running . also , incorporate a sufficient number of days for recovery . ice application : apply ice to the injured area for 30 minutes twice a day . finally , in a case series done at the stanford sports medicine clinic , 24 runners ( 10 m , 14 w ) with itbs completed a 6-week rehabilitation program , which consisted of local application of ultrasound with corticosteroid gel for the first two sessions . all patients were instructed to stretch the it band three times a day , and hip abduction exercises and pelvic drop sets to strengthen the gluteus were increased throughout the program with a goal of 3 sets of 30 repetitions . nonsteroidal anti - inflammatory drugs were prescribed until the patients were free of pain during daily activities . the investigators found a mean increase of 34.9% and 51.4% in the injured leg of the hip abductor torque for females and males , respectively . twenty - two of the 24 athletes ( 91.7% ) of the athletes were able to return to running at the end of the 6-week program . these four studies demonstrate how diverse conservative treatment for itbs can be . from clinical experience this treatment becomes less useful as it becomes a more chronic condition when bursal and periosteal changes have set in . there is limited evidence to support one specific approach to the treatment of itbs ; however , when looking at the desired goal of return to sport , a combination of rest ( 26 weeks ) , stretching , pain management , and modification of running habits produces a high return to sport rate . surgery is often reserved for refractory cases that have failed other avenues of conservative management . however , in the athletic population , return to sport is a common concern , and multiple , long absences from sport due to trials of various conservative treatment approaches are often not ideal . suggests that conservative treatments should be maintained for an average of 9 months before consideration of surgical intervention . others have based their decision for surgical intervention on the observation that at 30 degrees flexion , the posterior fibers of the itb are tighter against the lateral femoral epicondyle than are the more anterior fibers , in which case a surgical release in the posterior fibers is needed to correct the problem [ 14 , 15 ] . in one study , 36 athletes with a resistant itbs were treated with a standard arthroscopic technique , limited to the resection of lateral synovial recess . the patients had suffered from itbs for an average of 18 months ( 17 years ) . thirty - three patients ( mean age 31.1 years ) were available for followup at least 6 months postoperatively . prior to surgery , all patients had been treated conservatively for at least 6 months with rest , correction of training error , shoe modification , physical therapy and local infiltration with steroids . thirty - two patients had good or excellent results based on subjective functional results at followup . all patients went back to sports after 3 months . in 2 patients a meniscal lesion was found , which required treatment , and an associated cartilage lesion of the femoral condyle was found in the one patient that reported a fair outcome at followup . the author concluded that arthroscopic resection of the lateral synovial recess in resistant itbs is a valid option with a consistently good outcome , which also allows excluding or treating other intra - articular pathology . a retrospective study on athletes in norway looked at 45 patients who failed conservative management of itbs . the surgical procedure of choice for these resistant itbs cases was transection of the posterior half of the iliotibial band where it passes over the lateral epicondyle of the femur . with a mean age of 27 ( 1446 ) years , 38 ( 84.4% ) had excellent or good results , 6 ( 13.3% ) had fair results , and 1 ( 2.3% ) had a poor result . return to sport was not documented in this study , but 75.6% of patients reported that they would have the operation again . bursectomy has also been explored as a surgical treatment option for itbs . in a recent study a single surgeon performed 11 open iliotibial band bursectomies on 11 patients ( 7 m , 4 w ) . each patient presented with persistent ( > 6 months ) symptoms despite conservative treatment , with an average age at onset of 29 ( 2441 ) years . after a minimum of 20-month followup , all patients were able to return to their preinjury tegner activity levels , and all reported less pain ( 11-point visual analogue scale score decreased by 6 points ) . nine of the 11 patients said that knowing what they know now they would have the surgery performed again for the same problem . this population , however , was a mix of athletic and the general population , and the study did not separate out the results of each population . iliotibial band syndrome ( also called iliotibial band friction syndrome ) is a common problem encountered in the knees ' of athletes , especially endurance athletes whose sport requires repetitive knee flexion . itbs can often recur in the athletic population , causing significant morbidity and delay in return to sport . there is debate on whether iliotibial band syndrome is truly a friction syndrome where the itb itself is pathologic or whether a pathologic bursa forms between the itb and the lateral femoral condyle , causing the pain . this is an important concept because successful surgical treatment of the syndrome must address the underlying pathological causes . regardless of stance on the pathophysiology , conservative management is the first line of therapy for itbs . however , both conservative and surgical therapies play a major role in recalcitrant cases . a combination of therapies ( rest , pain relief , stretching , strength training , and running habit modification ) works best for returning athletes to their preinjury level and reducing their symptoms . however , a systematic regimen involving all aspects of conservative therapy has not been established . a recent systematic review on iliotibial band syndrome in runners concluded that there is limited evidence to support one specific approach to the diagnosis and treatment of itbs , suggesting that additional research is needed to elucidate an optimal treatment regimen . in our review , conservative therapy alone was found to have a 44% complete cure rate with return to sport at 8 weeks and a 91.7% return to sport rate at 6 months [ 2 , 12 ] . most surgeons who have published in the literature ascribe to the iliotibial friction band syndrome theory , and numerous procedures that excise or release this supposedly pathologic portion of the itb have been described . cortisone injections should still be used first in these scenarios , as itbs is considered an inflammatory friction syndrome . however , as the duration of symptoms increases and conservative measures fail , surgical treatment may be needed for resolution of symptoms . three of these procedures have been described above , all in athletes , with a return to sport rate of 100% at an average of 3 months and 7 weeks . did not find a bursa in either the 6 cadaveric specimens they dissected or the 2 symptomatic patients on whom they performed an mri . in patients with iliotibial band syndrome mri findings of the itb can be normal . in a study of 16 patients with itbs , 31% had a discrete fluid collection medial to the itb , with a normal looking itb . however , it was hypothesized that this collection likely arose from chronic inflammation beneath the itb , resulting in the formation of a secondary or adventitious bursa rather than from the inflammation of an existing primary bursa . there are also those that ascribe to the theory that the itb itself is not pathological in patients with iliotibial band syndrome , but rather the pain and functional deficits are generated by a pathological bursa that forms beneath the itb due to compression of that underlying tissue rather than a friction mechanism . in the study performed by hariri et al . described above , they consistently found what appears to be an inflamed bursa underlying a benign - appearing itb . this study , however , was not done solely on the athletic population whereas the ones described which favor the friction theory of iliotibial band syndrome were . surgical intervention is often only utilized after patients have failed conservative management for itbs , making return to preinjury level a difficult task . although there are two theories on the pathophysiology of itbs , when looking at return to sport rate in the athletic population , resection of the lateral synovial recess , after failure of conservative therapy provides an excellent return to sport rate . iliotibial band syndrome is a common cause of lateral knee pain in the athlete , especially runners and other endurance athletes . both conservative and surgical approaches are viable treatment options , and both need to be considered during treatment planning . while the majority of cases resolve with conservative management , resistant cases are seen in many athletes , requiring surgical intervention . the cases that require surgical intervention are often chronic in nature , and it is important to recognize the duration of symptoms so that surgical treatment can be initiated early . despite many options for both surgical and conservative treatment , there has yet to be consensus on one standard of care . further research is needed to delineate the true pathology behind iliotibial band syndrome in athletes , as well as the optimal treatment regimen .

iliotibial band syndrome ( itbs ) is a common injury in runners and other long distance athletes with the best management options not clearly established . this review outlines both the conservative and surgical options for the treatment of iliotibial band syndrome in the athletic population . ten studies met the inclusion criteria by focusing on the athletic population in their discussion of the treatment for iliotibial band syndrome , both conservative and surgical . conservative management consisting of a combination of rest ( 26 weeks ) , stretching , pain management , and modification of running habits produced a 44% complete cure rate , with return to sport at 8 weeks and a 91.7% cure rate with return to sport at 6 months after injury . surgical therapy , often only used for refractory cases , consisted of excision or release of the pathologic distal portion of the iliotibial band or bursectomy . those studies focusing on the excision or release of the pathologic distal portion of the iliotibial band showed a 100% return to sport rate at both 7 weeks and 3 months after injury . despite many options for both surgical and conservative treatment , there has yet to be consensus on one standard of care . certain treatments , both conservative and surgical , in our review are shown to be more effective than others ; however , further research is needed to delineate the true pathophysiology of iliotibial band syndrome in athletes , as well as the optimal treatment regimen .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusion

PMC4719319

stress fractures occur in individuals in whom repetitive strenuous muscle and tendon force act on bone ; that have not adapted to such forces . under a constant load , osteoclast resorption and osteoblastic reconstruction of bone are in equilibrium , resulting in normal remodeling . if loading increases , additional bone resorption occurs . increased osteoclastic activity at sites of stress may cause local weakening and predispose to micro damage . a 30-year - old man presented with right thigh pain for 3 days without any history of significant trauma . he was a military recruit with history of running 5 miles a day for last 12 years and was running 20 miles a day for last 5 days before he developed pain . here we have reported a case of stress fracture of proximal femur in intertrochanteric region which to our knowledge has not been reported in the literature so far . this fracture is important to recognize early as there are high chances of displacement resulting in increased risk of complications . we suggest immediate anatomical reduction and stable internal fixation to prevent complications and early mobilization to decrease the morbidity . stress fractures occur in individuals in whom repetitive strenuous muscle and tendon force act on bone ; that have not adapted to such forces.[1 - 3 ] stress fractures can be subdivided into fatigue fractures , caused when normal bone is exposed to repeated abnormal stress , and insufficiency fractures , where normal stress is applied to abnormal bone . under a constant load , osteoclast resorption and osteoblastic reconstruction of bone are in equilibrium , resulting in normal remodelling . if loading increases , additional bone resorption occurs . increased osteoclastic activity at sites of stress may cause local weakening and predispose to micro damage . although stress fractures can arise at any site , the most common locations are postero - medial tibia , particularly in runners ; metatarsals in runners , dancers , and military recruits ; iliopubic and ischiopubic rami in military recruits , gymnasts , dancers , and soccer players ; and femur in crosscountry runners . stress fractures of the femur are usually seen in osteoporotic elderly people , but in healthy young athletes they are less common . fractures localized to the superior surface of neck of femur are termed tension fractures , and those localized to the inferior surface , compression fractures . to our knowledge , the literature contains no reports of stress fracture of proximal femur in intertrochanteric region . here , we describe a case of stress fracture of the proximal femur in intertrochanteric region in a male military recruit . the patient was informed that data concerning the case would be submitted for publication , and he consented . a 30-year - old man presented with right thigh pain for 3 days without any history of significant trauma . he was a military recruit with history of running 5 miles a day for last 12 years and was running 20 miles a day for last 5 days before he developed pain . all blood investigations including serum levels of calcium , phosphorus , pth and creatinine were normal except for low 25-hydroxy vitamin d3 levels ( 22.4 ng / ml ) . reports of 99m - technetium mdp bone scan , magnetic resonance imaging ( mri ) and computed tomography ( ct ) were consistent with stress fracture with no other pathology . patient was managed by closed reduction and internal fixation with dynamic hip screw with anti - rotation screw . initially patient was molilised toe touch walking with cruch support with regular follow up and at the present time patient is walking full weight wearing , no pain , without support and performing his daily routine activities and joined his duties as well . the literature renders different opinions and data with regard to stress fractures and the structural integrity of bones . a number of authors feel that wolff 's law is applicable with stress fractures in that the bone is attempting to adapt to the ongoing stresses . it appears that bone resorption with accelerated repetitive stress occurs at a greater rate than bone deposition . the end result is a stress fracture from repetitive , cumulative stress exceeding the structural strength of the bone . histologically , as humans mature from adolescence , the bone in the femoral neck undergoes internal remodeling of circumferential lamellar bone to adult osteonal bone . experimentally , the greater the percentage of osteones with lamellar bone , the greater the resistance to failure with repeated loading . fractures appear to be related to the rate of loading , if the rate of loading exceeds the rate of there modeling of bony buildup , then a fracture may occur with stress . the femoral neck is subjected to loading forces several times body weight and with stands considerable tensile and compressive forces . it is important to consider these two distinct forces separately because they lead to different injury types and outcomes . tensile forces occur at the superior aspect of the femoral neck , where as compressive forces occur at the inferior aspect . [ 12 - 14 ] other biomechanical factors , such as leg length inequality , coxavara , and pescavus , may also be important in the development of compressive and tensile injuries at the femoral neck . femoral neck stress fracture has many associated complications such as nonunion , malunion , osteonecrosis , and arthritic changes . many authors suggest that tension side femoral neck stress fractures require internal fixation because of potential instability and high rate of complications ; however , there are reports of successful conservative management for non - displaced tension side fractures compression side fractures are generally treated conservatively with a period of rest followed by gradual return to activity and exercises . femoral neck stress fractures treated conservatively should have frequent radiographic monitoring for progression given the high incidence . return to running is considered when full weight bearing is asymptomatic , there is no tenderness to palpation on physical examination , and imaging studies are consistent with healed fracture . literature is not available on stress fracture in pertrochanteric region so biomechanics related to it is not clear . the pertrochanteric region is quite variable in its combination of cortical and cancellous bone structure . the well - vascularized pertrochanteric region is dependent on the structural integrity of a laminated cancellous bone arcade from the femoral head and epiphyseal scar , around ward 's triangle to the lesser trochanter , where the solid nature of the structure changes to a tubular construct with the origin of the femoral medullary canal ; the strong plate of bone posteriorly is named the calcar femorale . this is the region most affected with the posteromedial fracture comminution leaving only the anteromedial cortex potentially stable . the main structural attachments to the proximal femur include the hip capsule and the musculotendinous junctions of the gluteus medius and minimus ( greater trochanter ) , iliopsoas ( lesser trochanter ) , pirifomis and short external rotators ( posterior trochanteric region from the greater trochanteric region to the lesser trochanter ) , the oblique head of the rectus femoris ( anterior capsule ) , and the vastus lateralis ( lateral femur distal to the greater trochanter ) . the hip capsule is especially important in reduction of pertrochanteric fractures and its continuity with the distal fragment is the soft tissue attachment on which a stable reduction is possible . with capsular disruption , the displacement of the fracture fragments is dependent on the musculotendinous attachment to the respective fragments . the greater trochanter is abducted and externally rotated by the gluteus medius and short external rotators , the shaft is displaced posteriorly and medially by the adductors and hamstrings . lambotte described the four components of surgical treatment of fractures at the turn of the twentieth century , and they are as applicable today as then . the first is exposure of the fracture , which today means visualization of the fracture deformity , and the safest approach to ensure reduction and placement of the implant in the correct position . the second is reduction of the fracture , which is critical to the stability and functional recovery of the patient . inadequate reduction is the major preventable etiology for lost reduction and implant failure in pertrochanteric fractures . the third step is provisional fixation in an anatomically reduced position ; this is frequently the most neglected step in hip fracture surgery . this involves the reduction of the fracture and then maintenance of the fracture with either provisional kirschner pins and/or clamps to hold the fracture in position while the bone is prepared for the definitive implant . the last step is definitive fixation , which should maintain the reduced fracture in an acceptable anatomic and functionally correct position until fracture healing is complete . [ lambotte principal of fracture management was basically given for traumatic fractures that is still followed for such injury . it means lambotte principal of fracture management can be applied for fracture caused by severe trauma and also for stress fracture . here we have reported a case of stress fracture of proximal femur in intertrochanteric region which to our knowledge has not been reported in the literature so far . this fracture is important to recognize early as there are high chances of displacement resulting in increased risk of complications . we suggest immediate anatomical reduction and stable internal fixation to decrease the morbidity and prevent complications . so any patient particularly military recruit if presented with mild pain in groin during activity and relieved by rest must be suspected for insufficiency fracture . if on plane radiography lesion is not visible , go with the most common investigation i.e. mri . if the fracture is displaced , it should be fixed to prevent complications and early mobilization to decrease morbidity of patient .

introduction : stress fractures occur in individuals in whom repetitive strenuous muscle and tendon force act on bone ; that have not adapted to such forces . under a constant load , osteoclast resorption and osteoblastic reconstruction of bone are in equilibrium , resulting in normal remodeling . if loading increases , additional bone resorption occurs . increased osteoclastic activity at sites of stress may cause local weakening and predispose to micro damage . if allowed to progress , such micro fractures may progress to complete fractures.case report : a 30-year - old man presented with right thigh pain for 3 days without any history of significant trauma . he was a military recruit with history of running 5 miles a day for last 12 years and was running 20 miles a day for last 5 days before he developed pain . examination revealed pain to palpation along the proximal medial and lateral right thigh . range of motion was painful and limited . radiograph of right hip showed fracture line in intertrochanteric region of femur.conclusion:here we have reported a case of stress fracture of proximal femur in intertrochanteric region which to our knowledge has not been reported in the literature so far . this fracture is important to recognize early as there are high chances of displacement resulting in increased risk of complications . we suggest immediate anatomical reduction and stable internal fixation to prevent complications and early mobilization to decrease the morbidity .

Introduction: Case Report: Conclusion: Introduction Case Report Discussion Conclusion

PMC4817897

standard techniques were used for the preparation of frozen sections ; hematoxylin and eosin ( h&e ) , gomori trichrome , nadh - tetrazolium reductase ( tr ) , and atpase staining ; and immunofluorescent and electron microscopic analysis . mouse monoclonal antibody against stim1 and rabbit polyclonal antibody against orai were obtained from bd biosciences ( franklin lakes , nj ) and merck ( darmstadt , germany ) , respectively . exonic sequences were enriched using a sureselect v5+utrs kit ( agilent , santa clara , ca ) and subjected to nucleotide sequence analysis using a hiseq2500 platform ( illumina , san diego , ca ) . burrows - wheeler aligner and samtools were used with default settings for the alignment of raw reads and variant detection . the protein - altering variants in stim1 and orai1 were filtered by excluding those present in 600 healthy japanese controls . direct nucleotide sequence analysis was performed using an abi 3100 genetic analyzer ( thermo fisher scientific , waltham , ma ) . messenger rna ( mrna ) was extracted from muscle biopsy specimen using rneasy fibrous tissue mini kit ( qiagen , hilden , germany ) and amplified by reverse transcriptase ( rt)-pcr with superscript ( thermo fisher scientific ) . expression plasmid encoding human stim1 complementary dna was purchased from origene and the mutation c.1450_1451insga was introduced with a kod - plus mutagenesis kit ( toyobo , osaka , japan ) . two days after transfection using effectene ( qiagen ) , c2c12 myoblasts were labeled with anti - stim1 , alexa fluor 594conjugated phalloidin ( thermo fisher scientific ) and 4,6-diamidino-2-phenylindole ( dapi ) . the images were obtained using an fsx100 fluorescence microscope ( olympus , tokyo , japan ) . transfection was carried out using x - tremegene hp transfection reagent ( roche , basel , switzerland ) . transfected hek293 cells were trypsinized and seeded onto poly - l - lysine coated glass coverslips the next day . cells on coverslips were loaded with fura-2 by incubation in dulbecco 's modified eagle medium containing 5 m fura-2/am ( dojindo , kumamoto , japan ) at 37c for 40 minutes . the coverslips were then placed in a perfusion chamber mounted on the stage of a microscope ( axio - observer z1 ; carl zeiss , oberkochen , germany ) . fura-2 fluorescence images recorded at 10-second intervals were analyzed using physiology software ( carl zeiss ) . standard techniques were used for the preparation of frozen sections ; hematoxylin and eosin ( h&e ) , gomori trichrome , nadh - tetrazolium reductase ( tr ) , and atpase staining ; and immunofluorescent and electron microscopic analysis . mouse monoclonal antibody against stim1 and rabbit polyclonal antibody against orai were obtained from bd biosciences ( franklin lakes , nj ) and merck ( darmstadt , germany ) , respectively . exonic sequences were enriched using a sureselect v5+utrs kit ( agilent , santa clara , ca ) and subjected to nucleotide sequence analysis using a hiseq2500 platform ( illumina , san diego , ca ) . burrows - wheeler aligner and samtools were used with default settings for the alignment of raw reads and variant detection . the protein - altering variants in stim1 and orai1 were filtered by excluding those present in 600 healthy japanese controls . direct nucleotide sequence analysis was performed using an abi 3100 genetic analyzer ( thermo fisher scientific , waltham , ma ) . messenger rna ( mrna ) was extracted from muscle biopsy specimen using rneasy fibrous tissue mini kit ( qiagen , hilden , germany ) and amplified by reverse transcriptase ( rt)-pcr with superscript ( thermo fisher scientific ) . expression plasmid encoding human stim1 complementary dna was purchased from origene and the mutation c.1450_1451insga was introduced with a kod - plus mutagenesis kit ( toyobo , osaka , japan ) . two days after transfection using effectene ( qiagen ) , c2c12 myoblasts were labeled with anti - stim1 , alexa fluor 594conjugated phalloidin ( thermo fisher scientific ) and 4,6-diamidino-2-phenylindole ( dapi ) . the images were obtained using an fsx100 fluorescence microscope ( olympus , tokyo , japan ) . transfection was carried out using x - tremegene hp transfection reagent ( roche , basel , switzerland ) . transfected hek293 cells were trypsinized and seeded onto poly - l - lysine coated glass coverslips the next day . cells on coverslips were loaded with fura-2 by incubation in dulbecco 's modified eagle medium containing 5 m fura-2/am ( dojindo , kumamoto , japan ) at 37c for 40 minutes . the coverslips were then placed in a perfusion chamber mounted on the stage of a microscope ( axio - observer z1 ; carl zeiss , oberkochen , germany ) . fura-2 fluorescence images recorded at 10-second intervals were analyzed using physiology software ( carl zeiss ) . he first noticed slight difficulty walking when he was 37 years old . at the age of 40 , running and climbing stairs became difficult . his mother also had difficulty walking and was in a wheelchair when she was 40 years old . his parents were not consanguineous . on neurologic examination , pupils and ocular movement were normal . muscle atrophy and weakness was evident in all 4 limbs , and the bilateral triceps surae muscles were most prominently affected ( figure 1a ) . the paraspinal muscles were also atrophic and he had winged scapulae ( figure 1b ) . he did not exhibit myalgia , cramps , scoliosis , rigid spine , or symptoms suggesting stormorken syndrome , including miosis , thrombocytopenia , asplenia , or ichthyosis . blood cell counts and routine blood biochemistry were normal , except serum creatine kinase was elevated to 798 iu / l ( normal range < 150 ) . muscle mri revealed disseminated high - intensity areas on t1-weighted images in the quadriceps femoris , triceps surae , and paraspinal muscles , indicating fatty replacement in these muscles ( figure 1c ) . ( a ) muscle atrophy is evident in all 4 limbs . among them , the triceps surae muscle ( white arrowhead ) is most prominently affected . ( b ) the patient exhibits winged scapulae due to atrophy of the paraspinal muscles . ( c ) muscle mri shows high - intensity areas on t1-weighted images in the quadriceps femoris , triceps surae , and paraspinal muscles , indicating fatty replacement in these muscles . ( d ) hematoxylin and eosin ( h&e ) staining reveals the increased variability of muscle fiber diameter and occasional centrally nucleated fibers . many fibers show tubular aggregates that are stained dark red by h&e ( d ) , intense blue by nadh - tetrazolium reductase ( e ) , and bright purple with gomori trichrome staining ( f ) . ( g ) a serial section stained with atpase ( ph 4.6 ) demonstrates that tubular aggregates are in type 2 fibers . ( h ) electron microscopy shows that some areas in the myofiber are replaced by numerous tubular structures . at higher magnification , each tubule is arranged in a honeycomb - like structure showing double - walled membranes ( inset ) . bar = 1 m . ps = paraspinal muscles ; qf = quadriceps femoris ; ts = triceps surae . in addition , many fibers showed irregularly shaped dark red staining ( figure 1d ) . these dark red accumulations were stained intense blue by nadh - tr ( figure 1e ) and bright purple with gomori trichrome staining ( figure 1f ) , indicating that these are tubular aggregates . a serial section stained with atpase ( ph 4.6 ) further demonstrated that the tubular aggregates were in type 2 fibers ( figure 1 g ) . electron microscopic analysis revealed that some areas in the myofiber were replaced by numerous tubular structures ( figure 1h ) and that each tubule was arranged in a honeycomb - like structure that showed double - walled membranes at higher magnification ( figure 1h , inset ) . exome sequence analysis and subsequent sanger sequencing of the proband revealed a novel heterozygous insertion mutation , c.1450_1451insga ( p.ile484argfsx21 ) , in exon 7 of stim1 ( figure 2a ) . the same mutation was identified in his mother but not in healthy family members ( figure 2b ) . the amino acid 484 resides in the ctid located in the cytoplasmic region of stim1 ( figure 2c ) . to assess the localization of stim1 in the skeletal muscle of the patient the immunolabeling of stim1 and orai1 , a calcium channel in plasma membrane , colocalized to the tubular aggregates visualized by gomori trichrome staining ( figure 2d ) , which is consistent with previous reports . because premature termination codons often result in nonsense - mediated decay of mrna , we analyzed mrna transcript in the muscle biopsy specimen . sequencing of the rt - pcr product demonstrated that both wild - type and the c.1450_1451insga mutant transcripts are present ( figure 2e ) . ( a ) sanger sequencing reveals a heterozygous insertion mutation , c.1450_1451insga , in stim1 . ( b ) pedigree of the family indicates dominant inheritance and sequence analysis demonstrates the segregation of the heterozygous mutations with the disease . the mutation p.ile484argfsx21 resides in the c - terminal inhibitory domain ( ctid ) , which is located in the cytoplasmic region of stim1 ( red type ) . all missense mutations reported so far reside in the ef hand in the luminal region ( blue type ) . ( d ) serial sections of gomori trichrome staining and immunofluorescent images indicate that both stim1 and orai1 colocalize to the tubular aggregates . ( e ) sanger sequencing of reverse transcriptase - pcr product amplified from muscle biopsy specimen demonstrates that both wild - type and mutant transcripts are present . cc1 = coiled - coil region 1 ; k = polylysine ; s = signal peptide ; sam = sterile motif ; sore = stim1 orai activating region ; sp = serine- and proline - rich domain ; tm = transmembrane domain . to investigate the functional significance of the c.1450_1451insga mutation typically , wild - type stim1 displayed diffuse distribution in c2c12 cells ( figure 3a ) . in sharp contrast , mutant stim1 concentrated intensely around nuclei and exhibited an aggregation - like appearance in shrunk cytoplasm ( figure 3a ) . counting cells with the diffuse vs aggregation - like localization of stim1 confirmed that the majority of cells expressing wild - type stim1 showed diffuse localization whereas most cells expressing mutant stim1 exhibited an aggregation - like pattern ( figure 3b ) . ( a ) c2c12 myoblasts were transfected with wild - type ( wt ) and the c - terminal inhibitory domain mutant stim1 and labeled with anti - stim1 antibody , phalloidin , and 4',6-diamidino-2-phenylindole ( dapi ) . wt stim1 displays diffuse distribution in c2c12 cells , whereas the signal of the mutant stim1 concentrates intensely around nuclei and exhibits an aggregation - like appearance in the shrunk cytoplasm . ( b ) seven stitching images ( 5 5 ) of the c2c12 myoblasts were captured using a fluorescence microscope , and the number of cells with diffuse vs aggregation - like localization of stim1 was counted . the majority of cells expressing wt stim1 show diffuse localization , whereas most cells expressing the mutant stim1 exhibit the aggregation - like pattern . ( c ) intracellular ca concentration was measured in hek293 cells expressing wt stim1 ( gray square ; n = 40 cells ) , the mutant stim1 ( black triangle ; n = 42 cells ) , or control ( open circle ; n = 47 cells ) . the perfusion solution was changed from hepes - buffered saline ( hbs ) containing 2 mm ca to ca - free solution containing 0.5 mm ethylene glycol tetraacetic acid ( egta ) . to evaluate ca influx across the plasma membrane , the duration of exposure to ca - containing hbs and ca - free hbs is indicated above the graph . ( d ) maximal intracellular ca rises after readdition of 2 mm ca to the cells . p = 0.0028 , 0.0104 , and 0.5299 for wt vs mutant , control vs wt , and control vs mutant , respectively . finally , we performed ca measurements to examine whether the stim1 mutation affects store - operated ca entry ( soce ) . the wild - type or the mutant stim1 was expressed in hek293 cells , and changes in the intracellular ca concentration ( [ ca]i ) were evaluated . as shown in figure 3c , overexpression of wild - type stim1 caused an increased [ ca]i in the perfusion solution containing 2 mm ca , suggesting that wild - type stim1 enhanced ca influx through orai1 endogenously expressed in hek293 cells . this notion was further confirmed by the fact that removal of the external ca by ethylene glycol tetraacetic acid led to decreased [ ca]i and that readdition of ca to the perfusion solution resumed the ca response . however , the mutant stim1-expressed cells did not show significant ca responses by the changes in the external ca concentration ( figure 3 , c and d ) . these results collectively suggest that the stim1 mutation we found in this study causes dysregulated ca homeostasis . he first noticed slight difficulty walking when he was 37 years old . at the age of 40 , running and climbing stairs became difficult . his mother also had difficulty walking and was in a wheelchair when she was 40 years old . his parents were not consanguineous . on neurologic examination , pupils and ocular movement were normal . muscle atrophy and weakness was evident in all 4 limbs , and the bilateral triceps surae muscles were most prominently affected ( figure 1a ) . the paraspinal muscles were also atrophic and he had winged scapulae ( figure 1b ) . he did not exhibit myalgia , cramps , scoliosis , rigid spine , or symptoms suggesting stormorken syndrome , including miosis , thrombocytopenia , asplenia , or ichthyosis . blood cell counts and routine blood biochemistry were normal , except serum creatine kinase was elevated to 798 iu / l ( normal range < 150 ) . muscle mri revealed disseminated high - intensity areas on t1-weighted images in the quadriceps femoris , triceps surae , and paraspinal muscles , indicating fatty replacement in these muscles ( figure 1c ) . ( a ) muscle atrophy is evident in all 4 limbs . among them , the triceps surae muscle ( white arrowhead ) is most prominently affected . ( b ) the patient exhibits winged scapulae due to atrophy of the paraspinal muscles . ( c ) muscle mri shows high - intensity areas on t1-weighted images in the quadriceps femoris , triceps surae , and paraspinal muscles , indicating fatty replacement in these muscles . ( d ) hematoxylin and eosin ( h&e ) staining reveals the increased variability of muscle fiber diameter and occasional centrally nucleated fibers . many fibers show tubular aggregates that are stained dark red by h&e ( d ) , intense blue by nadh - tetrazolium reductase ( e ) , and bright purple with gomori trichrome staining ( f ) . ( g ) a serial section stained with atpase ( ph 4.6 ) demonstrates that tubular aggregates are in type 2 fibers . ( h ) electron microscopy shows that some areas in the myofiber are replaced by numerous tubular structures . at higher magnification , each tubule is arranged in a honeycomb - like structure showing double - walled membranes ( inset ) . bar = 1 m . ps = paraspinal muscles ; qf = quadriceps femoris ; ts = triceps surae . in addition , many fibers showed irregularly shaped dark red staining ( figure 1d ) . these dark red accumulations were stained intense blue by nadh - tr ( figure 1e ) and bright purple with gomori trichrome staining ( figure 1f ) , indicating that these are tubular aggregates . a serial section stained with atpase ( ph 4.6 ) further demonstrated that the tubular aggregates were in type 2 fibers ( figure 1 g ) . electron microscopic analysis revealed that some areas in the myofiber were replaced by numerous tubular structures ( figure 1h ) and that each tubule was arranged in a honeycomb - like structure that showed double - walled membranes at higher magnification ( figure 1h , inset ) . exome sequence analysis and subsequent sanger sequencing of the proband revealed a novel heterozygous insertion mutation , c.1450_1451insga ( p.ile484argfsx21 ) , in exon 7 of stim1 ( figure 2a ) . the same mutation was identified in his mother but not in healthy family members ( figure 2b ) . the amino acid 484 resides in the ctid located in the cytoplasmic region of stim1 ( figure 2c ) . to assess the localization of stim1 in the skeletal muscle of the patient the immunolabeling of stim1 and orai1 , a calcium channel in plasma membrane , colocalized to the tubular aggregates visualized by gomori trichrome staining ( figure 2d ) , which is consistent with previous reports . because premature termination codons often result in nonsense - mediated decay of mrna , we analyzed mrna transcript in the muscle biopsy specimen . sequencing of the rt - pcr product demonstrated that both wild - type and the c.1450_1451insga mutant transcripts are present ( figure 2e ) . ( a ) sanger sequencing reveals a heterozygous insertion mutation , c.1450_1451insga , in stim1 . ( b ) pedigree of the family indicates dominant inheritance and sequence analysis demonstrates the segregation of the heterozygous mutations with the disease . the mutation p.ile484argfsx21 resides in the c - terminal inhibitory domain ( ctid ) , which is located in the cytoplasmic region of stim1 ( red type ) . all missense mutations reported so far reside in the ef hand in the luminal region ( blue type ) . ( d ) serial sections of gomori trichrome staining and immunofluorescent images indicate that both stim1 and orai1 colocalize to the tubular aggregates . ( e ) sanger sequencing of reverse transcriptase - pcr product amplified from muscle biopsy specimen demonstrates that both wild - type and mutant transcripts are present . cc1 = coiled - coil region 1 ; k = polylysine ; s = signal peptide ; sam = sterile motif ; sore = stim1 orai activating region ; sp = serine- and proline - rich domain ; tm = transmembrane domain . to investigate the functional significance of the c.1450_1451insga mutation , we transfected c2c12 myoblasts with wild - type and mutant stim1 . typically , wild - type stim1 displayed diffuse distribution in c2c12 cells ( figure 3a ) . in sharp contrast , mutant stim1 concentrated intensely around nuclei and exhibited an aggregation - like appearance in shrunk cytoplasm ( figure 3a ) . counting cells with the diffuse vs aggregation - like localization of stim1 confirmed that the majority of cells expressing wild - type stim1 showed diffuse localization whereas most cells expressing mutant stim1 exhibited an aggregation - like pattern ( figure 3b ) . ( a ) c2c12 myoblasts were transfected with wild - type ( wt ) and the c - terminal inhibitory domain mutant stim1 and labeled with anti - stim1 antibody , phalloidin , and 4',6-diamidino-2-phenylindole ( dapi ) . wt stim1 displays diffuse distribution in c2c12 cells , whereas the signal of the mutant stim1 concentrates intensely around nuclei and exhibits an aggregation - like appearance in the shrunk cytoplasm . ( b ) seven stitching images ( 5 5 ) of the c2c12 myoblasts were captured using a fluorescence microscope , and the number of cells with diffuse vs aggregation - like localization of stim1 was counted . the majority of cells expressing wt stim1 show diffuse localization , whereas most cells expressing the mutant stim1 exhibit the aggregation - like pattern . ( c ) intracellular ca concentration was measured in hek293 cells expressing wt stim1 ( gray square ; n = 40 cells ) , the mutant stim1 ( black triangle ; n = 42 cells ) , or control ( open circle ; n = 47 cells ) . the perfusion solution was changed from hepes - buffered saline ( hbs ) containing 2 mm ca to ca - free solution containing 0.5 mm ethylene glycol tetraacetic acid ( egta ) . to evaluate ca influx across the plasma membrane , 2 mm ca was applied to the cells . the duration of exposure to ca - containing hbs and ca - free hbs is indicated above the graph . ( d ) maximal intracellular ca rises after readdition of 2 mm ca to the cells . p = 0.0028 , 0.0104 , and 0.5299 for wt vs mutant , control vs wt , and control vs mutant , respectively . finally , we performed ca measurements to examine whether the stim1 mutation affects store - operated ca entry ( soce ) . the wild - type or the mutant stim1 was expressed in hek293 cells , and changes in the intracellular ca concentration ( [ ca]i ) were evaluated . as shown in figure 3c , overexpression of wild - type stim1 caused an increased [ ca]i in the perfusion solution containing 2 mm ca , suggesting that wild - type stim1 enhanced ca influx through orai1 endogenously expressed in hek293 cells . this notion was further confirmed by the fact that removal of the external ca by ethylene glycol tetraacetic acid led to decreased [ ca]i and that readdition of ca to the perfusion solution resumed the ca response . however , the mutant stim1-expressed cells did not show significant ca responses by the changes in the external ca concentration ( figure 3 , c and d ) . these results collectively suggest that the stim1 mutation we found in this study causes dysregulated ca homeostasis . in the present study , we described a family with dominant tam with a novel frameshift mutation in the ctid of stim1 . of note , this is in contrast to the previous reports of tam , in which the proximal muscles were predominantly affected . in skeletal muscle , ca homeostasis is regulated by soce . in soce , stim1 forms an oligomer upon the depletion of ca in sr and activates orai1 channels at sarcolemma , triggering extracellular ca entry . all stim1 mutations in tam reported so far are missense mutations residing in the ef hand ( ef1 and ef2 in figure 2b ) , and this region is considered a hotspot . these mutations are presumed to induce the constitutive activation of orai1 and cause excessive ca influx into muscle cells . in contrast , a frameshift mutation , which creates a premature stop codon in the ctid located in the c - terminal cytoplasmic region of stim1 , was identified in our family ( figure 2b ) . the ctid is known to interact with soce - associated regulatory factor ( saraf ) , a regulator of slow ca - dependent inactivation of orai1 . following the transfection of c2c12 myoblasts with the ctid mutant stim1 , aggregation - like signals of stim1 the aggregation - like appearance is distinct from that in c2c12 myoblasts transfected with stim1 harboring ef hand mutations , which exhibit numerous puncta - like small clusters in cytoplasm . furthermore , intracellular ca measurements revealed that the ca influx is significantly decreased in the cells transfected with the ctid mutant compared to the wild - type stim1-transfected cells . this result is in marked contrast to the previous reports , in which increased ca influx caused by constitutive activation of soce was observed . the transcript of ctid mutant was present in the affected skeletal muscle of our patient , suggesting that nonsense - mediated mrna decay is not induced . we speculate that the ctid mutant may inhibit the oligomer formation of stim1 necessary to activate the orai1 channels , leading to the decreased intracellular ca influx . alternatively , the stim1 oligomer including the ctid mutant may not translocate properly to the subsarcolemma where the oligomer interacts with orai1 , because the ctid mutant lacks the polybasic motif ( k in figure 2c ) necessary for the stim1 targeting to plasma membrane . it is of particular interest to note that stim2 , an alternatively spliced form of stim2 , converts stim isoforms from an activator to an inhibitor of orai1 . our findings provide further evidence that the dysregulation of ca homeostasis underlies the pathomechanism of tam . dr . okuma contributed to the evaluation of patients , data acquisition interpretation and analysis , and manuscript preparation . saito contributed to the study concept and design , data acquisition , interpretation and analysis , and manuscript preparation . sonoo contributed to the study concept and design , data acquisition , interpretation , and analysis . takeda pharmaceutical co. ltd . , and daiichi sankyo co. ltd . ; and was supported by an intramural research grant ( 26 - 8 ) for neurological and psychiatric disorders of ncnp from the ministry of health , labour and welfare of japan and a grant - in - aid for scientific research c ( 26461281 ) from the ministry of education , culture , sports , science and technology of japan . mitsui has received research support from the ministry of education , culture , sports , science and technology of japan ( grant - in - aid for scientific research ( c ) 15k09334 ) . hara was supported by an intramural research grant ( 26 - 8 ) for neurological and psychiatric disorders of ncnp from the ministry of health , labour and welfare of japan , jsps kakenhi grant number 15h04846 , and a grant - in - aid for challenging exploratory research ( 25670149 , 15k13741 ) from the ministry of education , culture , sports , science and technology , japan ; and has received research support from the takeda science foundation , the life science foundation of japan , the kowa life science foundation , and the salt science research foundation . t. shimizu was supported by a grant - in - aid for scientific research c ( 15k09328 ) from the ministry of education , culture , sports , science and technology of japan . matsumura was supported by a grant - in - aid for scientific research c ( 25430075 ) from the ministry of education , culture , sports , science and technology of japan . j. shimizu was supported by a health and labour sciences research grant on intractable diseases ( evidence - based early diagnosis and treatment strategies for neuroimmunological diseases ) from the ministry of health , labour and welfare of japan , a grant - in - aid for scientific research c ( 24591289 ) from the ministry of education , culture , sports , science and technology of japan , and an intramural research grant ( 26 - 8 ) for neurological and psychiatric disorders of ncnp from the ministry of health , labour and welfare of japan . japan blood products organizations , mitsubishi tanabe pharma co. , bayer japan , janssen pharmaceutical , genzyme japan , eisai co. ltd . , fp - pharmaceutical co. , dainippon sumitomo pharma co. ltd . , otsuka pharmaceutical co. ltd . , sony corporation , nikkei business publications inc . , and leave a nest co. ltd . ; has served on the editorial board of neurology and clinical neuroscience ; has received research support from sanofi , japan blood products organization , mitsubishi tanabe pharma co. , pfizer japan inc . , ono pharmaceutical co. ltd . , daiichi sankyo co. ltd . , eisai co. ltd . , kowa pharmaceutical co. ltd . , and glaxosmithkline ; and was supported by kakenhi ( grant - in - aid for scientific research on innovative areas [ 22129001 and 22129002 ] ) , the japan agency for medical research and development ( grant - in - aid [ h26-nanchiseishikkan jitsuyoka - ippan-080 , 26310101 , and ninchisho kenkyu 26340101 ] ) , and a grant - in - aid ( h23-jitsuyoka [ nanbyo]-ippan-004 ) of the research on measures for intractable diseases from the ministry of health , welfare and labour , japan . sonoo has served on the editorial boards of muscle and nerve , clinical neurology , and the japanese journal of clinical neurophysiology ; and was supported by grants - in - aid for scientific research from the ministry of education , science , sports and culture of japan ( 23591285 ) , and a health and labour sciences research grant on rare and intractable diseases ( evidence - based early diagnosis and treatment strategies for neuroimmunological diseases ) from the ministry of health , labour and welfare of japan .

objective : to identify the gene mutation of tubular aggregate myopathy ( tam ) and gain mechanistic insight into the pathogenesis of the disorder.methods:we described a family affected by autosomal dominant tam and performed exome and sanger sequencing to identify mutations . we further analyzed the functional significance of the identified mutation by expression studies and intracellular ca2 + measurements.results:a 42-year - old man presented with slowly progressive muscle weakness and atrophy in all 4 limbs and the trunk . muscle biopsy and microscopic examination revealed tubular aggregates in his skeletal muscle . genetic analysis of this family identified a novel heterozygous mutation , c.1450_1451insga ( p.ile484argfsx21 ) , in stromal interaction molecule 1 ( stim1 ) , a ca2 + sensor in sarcoplasmic reticulum . we transfected cultured cells with stim1 and demonstrated that the mutant stim1 exhibited aggregation - like appearance in shrunk cytoplasm . furthermore , we revealed that the intracellular ca2 + influx is decreased by the mutant stim1.conclusions:the novel mutation p.ile484argfsx21 is located in the cytoplasmic c - terminal inhibitory domain ( ctid ) of stim1 . however , all mutations reported so far in tam reside in the luminal n - terminal ef hand region . the aggregation - like appearance of stim1 and the decreased intracellular ca2 + influx in cells transfected with ctid mutant are in sharp contrast to these previous reports . taken together , these findings indicate that mutations of stim1 cause tam through the dysregulation of ca2 + homeostasis .

METHODS Standard protocol approvals, registrations, and patient consents. Histologic analyses. Genetic analysis. Expression study of Measurement of changes in intracellular Ca RESULTS Clinical data. Pathologic findings. Genetic analysis and localization of Expression of mutant STIM1 in C2C12 myoblasts. Effects of DISCUSSION AUTHOR CONTRIBUTIONS STUDY FUNDING DISCLOSURE

PMC3272559

a 61 year old man presented to our institution with a diagnosis of stage t3a prostate cancer . his serum prostate - specific antigen level was 5.3 ng / ml , and a transrectal biopsy demonstrated a gleason score of 3 + 4=7 in 20% to 80% of the cores from both lobes . a combination of suture ligation , bipolar cautery , harmonic scalpel , and holc were successfully used at various stages of the procedure to help achieve hemostasis and for ligation of the vas deferens . holc were used specifically for control of the lateral pedicle of the prostate during ralrp . we did not use holc at the bladder neck or the site of vesicourethral anastomosis . the vesicourethral anastomosis was achieved by using a single intracorporeal knot running 2 - 0 monocryl suture . also , the patient did not undergo wide dissection of the bladder neck during ralrp . pathology showed a gleason score of 3 + 4=7 with extracapsular extension , but negative lymph nodes , negative margins , no seminal vesicle invasion , and no angiolymphatic invasion . three months after surgery , he complained of dysuria and decreased force of the urine stream . he underwent retrograde cystourethrography in the office , which revealed a bladder neck contracture on the urethra ( fig . the cystourethroscopy demonstrated a normal - looking urinary sphincter but a very tight bladder neck . the bladder neck was successfully dilated , and visualization of the bladder revealed the presence of four holc . the four holc were floating in the bladder without stone formation and had migrated from the previous ralrp ( fig . , an indwelling urethral catheter was left for 7 days . at the 3 month follow - up after this dilatation , the patient was satisfied with his voiding habits and reported having a good urinary stream . with the recent surge in minimally invasive surgery ( robotic and laparoscopic assisted surgery ) , interest in hemostatic alternatives to suturing has increased . several automated suturing devices including endostitch ( autosuture , ussc , norwalk , ct , usa ) , lapraty clips ( ethicon , endosurgery inc , piscataway , nj , usa ) , and holc have been used during laparoscopic procedures . among them , holc are a commonly used device and have been shown to be safe and reliable for vascular control in laparoscopic procedures . during ralrp , holc have been used for ligation of the vasa deferentia , seminal vesicle arteries , and prostatic pedicles to prevent excessive electrocautery and possible injury to the neurovascular bundles . however , several limitations , in addition to the concern for clip migration , have become apparent . blumenthal et al . reported the first case of migration of holc into the vesicourethral anastomosis after ralrp . there have been some reports of intravesical migration of these clips causing urethral erosion , bladder neck contractures , and subsequent calculus formation [ 2 - 6 ] . in all those cases , however , holc lodged in the bladder neck area with stone formation . in our case , the holc were floating in the bladder without stone formation . of a case in which a metal clip had migrated into the urinary bladder after retropubic radical prostatectomy . it was hypothesized that inflammation had arisen around the urinary bladder or vesicourethral anastomosis and that this also involved the metal clip , which then eroded the bladder wall and eventually migrated into the bladder . also , in our case , the mechanism of migration of holc in the bladder is unclear . according to the hypothesis described above , we can suspect that the holc migrated into the bladder during the serial course . on the basis of these findings , we recommend minimizing the use of holc on tissue immediately adjacent to the anastomosis during ralrp , specifically , the vasa and seminal vesicles , and every effort should be made to retrieve any loose clips after the procedure . these clips are prone to migration and may cause , or significantly contribute to , bladder neck contracture formation after ralrp . migration of holc into the vesicourethral anastomosis should be considered in patients with symptoms consistent with bladder neck contracture after ralrp . in addition , in these cases , noncontrast computed tomography scan might be helpful to detect clips .

hem - o - lok clips ( weck surgical instruments , teleflex medical , durham , nc , usa ) are widely used in robot - assisted laparoscopic radical prostatectomy because of their easy application and secure clamping . to date , there have been some reports of intravesical migration of these clips causing urethral erosion , bladder neck contractures , and subsequent calculus formation . we report the first case of bladder migration of hem - o - lok clips without stone formation after robot - assisted laparoscopic radical prostatectomy . the hem - o - lok clips were found during urethral dilation with a guide wire for bladder neck contracture under cystourethroscopy . the hem - o - lok clips were floating in the bladder without stone formation and were removed by a cystoscopic procedure .

CASE REPORT DISCUSSION

PMC3569930

preterm infants are at a high risk of motor deficits in later life , with approximately fourteen percent of very preterm infants developing cerebral palsy ( cp ) , and up to forty percent of very preterm infants demonstrating mild motor deficits . the mechanisms underlying such motor impairments have not yet been fully elucidated , but have been related to a number of factors , including abnormal cerebral development ( particularly in sensorimotor regions ) , conditions such as periventricular leukomalacia , peri - intraventricular hemorrhage , and/or stressors in the neonatal intensive care unit ( nicu ) environment . new insights into the neural structures and mechanisms , underlying motor function in preterm - born infants , should help in the development of new diagnostic and prognostic tools and provide information on the immediate efficacy of early intervention therapies . the corpus callosum is vital for communicating and integrating motor and somatosensory information between the hemispheres , and for bimanual motor coordination and function . the maturation or structural development of the corpus callosum , that is , the organisation of axon fiber bundles and the degree of axonal myelination and microstructural integrity , can be studied using diffusion tensor imaging ( dti ) , particularly via the measures of fractional anisotropy ( fa ) and mean diffusivity ( md ) . the interpretation of fa and md values is not entirely clear and largely depends on the underlying fiber architecture , including the degree of myelination , axon size , density , and organization [ 7 , 8 ] . since the corpus callosum is a highly organized myelinated structure , with axons typically running in packed parallel bundles , a high fa value and low md value is suggestive of greater structural maturation . in imaging studies , the infant corpus callosum is often segmented into six anatomical regions from anterior to posterior the genu , rostral body , anterior midbody , posterior midbody , isthmus , and splenium . thompson et al . found that very preterm infants had significantly reduced corpus callosum cross - sectional area , lower fa and higher md values compared to term infants , thus suggesting that corpus callosum development is altered in preterm - born infants compared to term - born infants . a minority of studies have investigated the implications of this altered development by examining associations between corpus callosum maturation and neurodevelopment [ 1113 ] investigated the relationship between dti measures of the genu and splenium ( the most anterior and posterior subregions of the corpus callosum , resp . ) taken at term age with neurological assessments ( amiel - tison scale , gross motor function classification system , and bayley scales of infant development ) performed at eighteen months in preterm - born children . the maturation of the splenium ( as reflected by fa values ) was significantly lower in children with abnormal versus normal neurological assessments , indicating that splenium fa near term age could potentially be used to prognosticate subsequent neurodevelopment . 2002 study using dti to examine white matter tracts in children with cerebral palsy as a result of periventricular leukomalacia . the tracts in the splenium , posterior corona radiate , and posterior internal capsule were markedly reduced in size in participants suffering from cerebral palsy compared to controls . hence , it was suggested that interhemispheric tracts to or from the sensory cortex , in addition to motor tracts , may also play a role in motor impairment . a strong positive association between the midsagittal surface area of the corpus callosum on t1-weighted magnetic resonance imaging ( mri ) scans and concomitant motor function in preterm - born children , studied at school age . furthermore , iai et al . compared midsagittal corpus callosum t1-weighted mri measures in preterm - born children with spastic diplegia ( cp ) versus neurologically typical children . in the diplegic children , it was found that the ratios of the thickness of the splenium and of the midbody to the corpus callosum length were significantly reduced , compared to the ratios computed in neurologically typical children . furthermore , the ratio for the splenium was highly correlated with the extent of motor impairment . however , aside from these few studies , potential associations between corpus callosum structure and neuro - motor function in preterm - born neonates have not been reported . outcomes from such investigations may prove clinically meaningful ; for example , they may inform development of early prognostic indicators , interventions , and/or markers to inform instigation of such interventions . hence the primary aim of this study was to investigate and explore potential associations between dti measures of subregions of the corpus callosum and concomitant neurological measures in a small sample of preterm infants . we hypothesized that a positive relationship would exist between corpus callosum maturation and concomitant neurological measures . the participants were preterm infants born at gestational age between 28 and 32 weeks ( ga ) at the royal brisbane and women 's hospital ( rbwh ) with no post - natal medical issues or complications . inclusion criteria were : infants that were born at a gestational age between 28 and 32 weeks , with a birth weight and length between the 10th and 90th percentiles for gestational age and who were determined to be medically stable . exclusion criteria were as follows : the presence of abnormalities on brain ultrasound ( i.e. , intraventricular hemorrhage grades 3 or 4 , persistent periventricular flares , or periventricular cysts ) and the presence of major genetic disorders or malformations . these criteria were set to ensure that the study participants were at low risk for an adverse outcome and were medically stable . this study was approved by the rbwh human research ethics committee and monash university human research ethics committee . parents who agreed to their infant participation in the study signed a written consent form and were provided with a copy of the study protocol . on reaching term - equivalent age ( 42 weeks ga ) , the infants underwent the hammersmith neonatal neurological examination ( hne ) at the rbwh performed by a single neonatologist . the hne evaluates a number of motor and behavioral functions including posture and tone , tone patterns , reflexes , abnormal signs and orientation , and behavior . when performed at around term age , it provides a summary score for each of these categories and a total hne score ( maximum score 32 ) which is the sum of these categories . as a follow - up analysis , a total motor - specific score ( maximum 20 ) was calculated by summing the scores from the posture and tone , tone patterns , and reflexes categories . associations between the hne and motor - specific scores and dti measures were examined . the infants underwent neuroimaging at approximately term - equivalent age ( refer to table 1 ) . the infants were scanned using a 3.0-t siemens tim trio scanner and were placed in an mri - compatible neonatal incubator with a dedicated head coil ( lammers medical , luebeck , germany ) . they were swaddled and placed in a vacuum fixation beanbag designed to keep the infant still and supported in the scanner . diffusion weighted images were acquired along 30 directions at a b - value of 1000 s / mm , along with one minimally diffusion weighted image ( b = 0 ) . the image resolution of the scans was 1.75 1.75 mm in - plane , with a slice thickness of 2 mm . other imaging parameters were as follows : tr / te 9300/130 ms , field of view 128 128 , 47 slices . the mri parameters for the field map acquired were tr / te1/te2 488/4.9/7.4 ms , matrix size 64 64 , field of view 160 160 mm , slice thickness 2.6 mm with 0.65 mm slice gap , 29 slices , and flip angle 60 degrees . preprocessing of the imaging data involved correction for any head movement using rigid - body registration with subsequent adjustment of the b - matrix , skull stripping , as well as susceptibility distortion correction and correction for intensity inhomogeneities . additionally , images were visually examined for artifacts and distortions and infants whose images showed motion artifact on the minimally diffusion - weighted image were excluded from the dti analyses . fractional anisotropy ( fa ) , color fa , and md maps of the brain were calculated for each infant , with fa representing the degree of anisotropic diffusion and md representing the magnitude of diffusion . alignment of images with the midsagittal plane was achieved using rigid - body registration of fa maps with the john hopkins university neonatal fa template . care was taken in the manual delineation of the corpus callosum in order to minimize the number of voxels that were contaminated by partial volume effects . for each infant , based on visual inspection , a corpus callosum region of interest was manually drawn onto the midsagittal slice of the color - fa maps . four sets of masks were drawn for each infant , to ensure reproducibility of the masks . for each infant , the four corpus callosum masks were then segmented into six regions genu , rostral body , anterior midbody , posterior midbody , isthmus , and splenium according to the neonatal segmentation schema provided by thompson et al . an average was taken of the mean values of the regions from the four masks to provide six corpus callosum fa and md values for each infant . the infants ranged between 41 and 45 weeks ga at the time of dti assessment . as it is known that fa values tend to increase rapidly with increasing ga , the fa values were adjusted for ga . using spss ( statistical package for the social sciences , version 14.0 ; ibm spss , chicago , il , usa ) , the correlations between clinical measures and corpus callosum dti measures were explored using a two - tailed spearman 's rho ( see tables 2 and 3 for the fa and md values used in the analysis ) . correction for multiple comparisons was not performed given the exploratory nature of this study of a small sample as well as the number and nature of the variables analyzed . table 1 provides a summary of the characteristics and assessments for these thirteen infants . due to time constraints furthermore , due to technical issues and motion artifacts , dti data from three infants was excluded from the analyses , leaving eight infants whose data was included in the correlation analyses . using spearman 's rho , associations were explored between the clinical and dti measures ( see tables 4 and 5 ) . a highly significant positive correlation was found between the total hne scores and the anterior midbody fa measures ( rho = 0.929 ; p = 0.001 ) and similarly between the motor - specific scores and the anterior midbody fa measures ( rho = 0.857 ; p = 0.007 ) . a statistically significant negative correlation was found between the total hne scores and the anterior midbody md measures ( rho = 0.714 ; p = 0.047 ) . scatter plots illustrating these correlations are provided in figures 2 , 3 , and 4 . in summary , the total hne scores correlated significantly with both the anterior midbody fa and md measures . in addition a significant positive correlation was found between the motor - specific subscores of the hne assessment and anterior midbody fa measures . overall neurological function , as well as specific motor function measures , correlated significantly with dti measures from the anterior midbody , but not from other regions of the corpus callosum . these findings are consistent with studies investigating corpus callosum topography in primates and humans [ 26 , 27 ] . the results from these studies suggest that , from a neuroanatomical perspective , axons in the midbody interconnect the premotor and primary motor cortical areas . furthermore , the primary and secondary motor cortices and somatosensory regions are located in the precentral and postcentral cortices , which are in a similar spatial region to the corpus callosum midbody . given that these somatosensory and motor regions , as well as the corpus callosum , are topographically organized , it is likely that interhemispheric axons which connect homologous regions of the left and right cortical regions pass through the corpus callosum midbody . in particular , these study findings suggest that such axons connecting ( frontal ) motor cortical regions likely constitute part of the anterior midbody . both total hne and motor - specific scores correlated with greater maturation of the anterior midbody ( i.e. , higher fa and lower md values ) . it has been suggested that fa values in the corpus callosum are primarily a reflection of fiber density and organization , rather than myelination or axon diameter in contrast , md values are believed to be more sensitive to ( pre-)myelination , with lower md values indicating advanced callosal myelination and microstructure [ 30 , 31 ] . consequently , a relatively well - organized and ( pre-)myelinated corpus callosum ( e.g. , anterior midbody ) may be associated with more efficient transmission of interhemispheric neural signals , which thereby permits more optimal processing and integration of ( e.g. , motor - related ) information between the hemispheres . this study appears to be one of the first studies to describe a strong correlation between the maturation of the anterior midbody and concomitant neurological function in preterm infants . in contrast , a number of studies ( as described in section 4 ) demonstrated associations between splenium maturation and altered neurological function . however , the infant cohorts used in these studies involved infants with demonstrated white - matter injury and periventricular leukomalacia ( pvl ) . a characteristic feature of pvl is thinning or altered maturation of the splenium ; hence , this may explain why a strong association was found between splenium maturation and altered neurological function in these infants . in light of these findings , intervention programs and management strategies directed towards enhancing the development of white matter should be investigated further , as this may aid in enhancing motor function and possibly other neurobehavioral functions for preterm infants . an mri study performed by smith et al . demonstrated that stresses from the nicu environment correlated with decreased white matter maturation and interhemispheric communication in preterm infants , as assessed by dti and functional mri . this study also reported that infants experiencing greater nicu - induced stress in the immediate postnatal period demonstrated poorer motor function at term - equivalent age . als et al . performed a randomized control trial investigating the merits of a newborn individualized developmental care and assessment program ( nidcap ) in order to reduce stressors for preterm infants while in nicu . they found improved white matter development ( assessed via dti ) , brain functional connectivity ( as measured by electroencephalography ) , and neurological function in the nidcap cohort of infants versus controls at term - equivalent age . a similar study performed by mcanulty et al . demonstrated that nidcap intervention had positive effects on preterm - born children 's neurobehavioral function at least until school age . since the current findings indicate an association between anterior midbody development and neurological function , this may in part explain why interventions such as the nidcap , which are directed towards enhancing white matter development , resulted in improved neuromotor function . the observed relationship between anterior midbody integrity and neurological function , if replicated and extended in larger studies , may inform future development of prognostic tools for neonates . future studies should investigate whether such dti measures may demonstrate value in the prediction of neurodevelopmental , particularly motor outcomes at a later age . conversely , in light of the strong correlation between hne scores and dti measures , it is possible that hne assessment may also prove to be a valuable prognostic marker , with the added advantage of being easy to perform at the bedside . such markers might also ultimately prove informative for future interventional trial programs , for example for selection of inclusion criteria , or predicting or assessing response to a given intervention . there are a number of limitations in the current study , particularly the small sample size and associated low statistical power . in addition , the corpus callosum segmentation method employed was not necessarily optimal as current understanding of corpus callosum anatomical segmentation and tract topography in infants is limited , unlike in adult studies . this would be likely improved by identification of callosal regions defined by dti tractography methods in infants . however a positive association was observed between dti measures of structural maturation of the anterior midbody of the corpus callosum and neurological and motor function , in healthy preterm - born infants assessed at term - equivalent age . future studies of larger samples may extend upon these preliminary findings and possibly investigate the value of corpus callosum dti measures as early prognostic markers for neurodevelopmental and motor outcomes .

background . the etiology of motor impairments in preterm infants is multifactorial and incompletely understood . whether corpus callosum development is related to impaired motor function is unclear . potential associations between motor - related measures and diffusion tensor imaging ( dti ) of the corpus callosum in preterm infants were explored . methods . eight very preterm infants ( gestational age of 2832 weeks ) underwent the hammersmith neonatal neurological examination and dti assessments at gestational age of 42 weeks . the total hammersmith score and a motor - specific score ( sum of hammersmith motor subcategories ) were calculated . six corpus callosum regions of interest were defined on the mid - sagittal dti slice genu , rostral body , anterior midbody , posterior midbody , isthmus , and splenium . the fractional anisotropy ( fa ) and mean diffusivity ( md ) of these regions were computed , and correlations between these and hammersmith measures were sought . results . anterior midbody fa measures correlated positively with total hammersmith ( rho = 0.929 , p = 0.001 ) and motor - specific scores ( rho = 0.857 , p = 0.007 ) . total hammersmith scores also negatively correlated with anterior midbody md measures ( rho = 0.714 , p = 0.047 ) . discussion . these results suggest the integrity of corpus callosum axons , particularly anterior midbody axons , is important in mediating neurological functions . greater callosal maturation was associated with greater motor function . corpus callosum dti may prove to be a valuable screening or prognostic marker .

1. Introduction 2. Participants and Methods 3. Results 4. Discussion 5. Conclusion

PMC3107436

fish is an important food item for the increasing world population which is rapidly approaching 7 billion people . fish , one of the main protein sources , is still predominantly caught from the wild . fishing operations are highly industrialized and effective , with remotely controlled nets , acoustic fish - finders and gps , resulting in severe depletion of stocks . aquaculture seems the obvious way out , but the fish farming industry currently meets questions regarding fish quality and environmental sustainability . the fishing industry , including fisheries and fish farming , is in fact targeting white muscle , the main part of fish lateral muscles , since it is preferred for human consumption ( johnston 1999 ) . many studies discuss the differences in quality between wild and farmed fish , see for example johnston et al . most fish have two separate muscular engines , one powered by red fibres for slow endured swimming and a much larger white one used for short fast bursts . adult fish use white muscles occasionally and only during short periods of time during fast escape or attack manoeuvres . unlike terrestrial animals , fish can afford to carry such a bulky specialised engine about because muscle tissue is virtually weightless under water and helps to streamline the body ( videler 1993 ) . cultivating fish for food requires knowledge of white muscle ontogeny , functional anatomy and the role in swimming , within the limits of the ecological niche of each species . novel ideas about improvement of cultivated fish quality must be based on fundamental knowledge about the natural development of the targeted tissue ( johnston 1999 ) . it is of prime importance for the fish food industry to understand what determines the number of white muscle fibres per unit cross - section . the development of white muscle tissue starts early during the embryonic phase and continues in newly hatched larvae . the fibre density in the white muscles is largely determined during the early phases of fish life . an overview of the structure , function , development and ecological role of white muscles under natural selection conditions will be compared with aquaculture practices to detect ways to improve white muscle properties in farmed fish . fish are aquatic vertebrates with a skull , a flexible but incompressible vertebral column supporting a medial septum dividing the body into two lateral halves , and lateral longitudinal muscles segmentally arranged in blocks , or myotomes between the sheets of collagenous myosepts . the myotomes are cone - shaped and stacked in a segmental arrangement on both sides of the median septum ( fig . the length of the cones varies between species . in the caudal region of mackerel ( scomber scombrus ) , cones span more than 5 vertebrae , in that of cod ( gadus morhua ) only 4 . in general , shorter segments and a larger number of cones per unit distance along the vertebral column increase the firmness of the lateral muscle.fig . 1a the myotomes and myosepts on the left side of the king salmon ( oncorhynchus tshawytscha ) . myotomes have been removed at four places to reveal the complex three - dimensional configuration of the lateral muscles . b a cross section through the upper left quarter of the caudal region of a salmon . the lines represent the myosepts between the complex myotomes ( based on shann 1914 ) a the myotomes and myosepts on the left side of the king salmon ( oncorhynchus tshawytscha ) . myotomes have been removed at four places to reveal the complex three - dimensional configuration of the lateral muscles . b a cross section through the upper left quarter of the caudal region of a salmon . the lines represent the myosepts between the complex myotomes ( based on shann 1914 ) in cross - sections through the caudal region , the muscles are arranged in four compartments . on each side , there is a dorsal and a ventral compartment , in some groups of fish separated by a horizontal septum . the left and right halves and the dorsal and ventral moieties are mirror images of each other . in cross - sections , the colour of the muscle fibres may be red , white or intermediate in different locations in the myotomes ( fig . white fibres form the bulk of lateral muscles , and in some species , intermediately coloured pink fibres are found between the two . in some fish , the colour differences are very obvious , but in others , the fibre type distribution is more confusing . the proportion of red muscle is highest in continuously active pelagic fish species ( i.e. 15% of the total muscle mass in mackerel , ) . ambush predators and other fish that spend most of the time motionless have predominantly white lateral muscles . they react to a single stimulus owing to the high density of nerve terminals on the fibres . the red colour is directly related to the stamina of these muscles because it is caused by an extensive blood supply with large amounts of the oxygen - binding protein myoglobin and high concentrations of large mitochondria , the cellular power plants . the red muscles of some large tuna and shark species are positioned well inside the white muscle mass , an arrangement which can increase the muscle temperature by as much as 10c during swimming ( carey and teal 1969 ) . this halves the twitch contraction time of the white muscles and doubles the maximum swimming speed . the diameter of red fibres is between 20 and 50% of that of white fibres where values can reach up to 300 m . it takes up to 24 h to remove lactic acid after a burst of all out activity . the high concentration of blood in red muscle tissue could be one reason . during the embryonic stage , the number of developing muscle cells depends on the temperature and oxygen levels ( johnston 2006 ) . the effects of decreased temperatures on the development of fast muscle fibres during this stage vary from no changes in numbers of muscle cells in sea bass ( dicentrarchus labrax ) , via fewer but larger cells in salmonids , turbot ( psetta maxima ) and plaice ( pleuronectes platessa ) , to more smaller cells in herring ( clupea harengus ) and cod . this variation implies that the effect of temperature on the numbers of white fibres at hatching needs to be investigated for each species separately . white muscle fibres start to develop during the early embryonic stages ( johnston et al . move vigorously within the confinement of the egg , exercising the white muscle fibres using short bursts of activity . at hatching , the lateral muscles of common carp consist of deep white muscle fibres covered with a monolayer of embryonic red fibres . after hatching , myogenesis continues by the increase in size and in numbers of the fibres present at hatching , see fig . 2 for the fibre arrangement of carp at first feeding . carp larvae have the highest growth rates when fed with zooplankton . during the first 2 weeks of feeding , the volume of trunk muscles increased more than 30-fold . hyperplasia ( the increase in cell numbers ) was responsible for 64% of this growth and hypertrophy ( the increase in cell size ) for 36% ( alami - durante et al . 1997).fig . 2schematic representation of one epaxial quadrant of a carp larva at first feeding showing the position of white muscle and the superficial layer of red muscle fibres . ( 1997 ) schematic representation of one epaxial quadrant of a carp larva at first feeding showing the position of white muscle and the superficial layer of red muscle fibres ( 1997 ) the increase in white muscle mass depends in a complex way on many environmental conditions , exercise is one of these . swimming in early larvae differs completely from that of larger stages , and early swimming movements are important for the development of white muscle . still inside the egg , fish larvae exercise the white muscle fibres making vigorous body movements . these may continue during the first hours after hatching and turn gradually into coordinated swimming movements after the yolk sac is absorbed ( mauguit et al . 2010 ) . during development from larva to adult , the nature of the interaction between fish and water changes from a viscosity - dominated regime during the early stages , to the flow regime mainly depends on the size and speed of the fish . the ratio of inertial to viscous forces is conveniently expressed by the dimensionless reynolds number . early fish larvae swim in a viscosity - dominated flow regime with reynolds numbers below 100 . for example , a 4-mm - long zebra danio ( brachydanio rerio ) larva can reach an initial velocity of 16 mm / s with an average acceleration of 200 mm / s . the active stroke takes 0.1 s , and the distance covered during the stroke is 1 mm . the initial speed rapidly drops due to the viscous mass of water dragged along by the coasting straight body . the total distance covered while coasting was 1 mm before the larva came to a halt after 1 s ( mller et al . 2000 ) . although the initial velocities reached are high relative to the body length of the larvae , they are very moderate in metres per second . distances covered during escape responses are extremely small , in the order of a few millimetres . this implies that escape from predators by swimming in an inertial regime is hardly possible . therefore , this vulnerable life phase is obviously very important for the development and growth of white muscle fibres . adult fish operate in inertia - dominated flow regimes with reynolds numbers well over 1,000 . small adults also often use a few tail beats followed by a gliding phase with the body stretched straight . an adult 35-mm - long zebra danio is able to accelerate at 1,600 mm / s to a speed of 130 mm / s using about the same time for the active stroke as the 4 mm larva . however , the subsequent gliding distance is 30 mm instead of 1 mm because the amount of water dragged along is much smaller in proportion to the size of the animal , and both the starting velocity and the inert accelerated mass are higher . this difference implies that the number of potential predators decreases with the size of fish . adult fish larger than 10 mm also swim by continuous lateral undulations of the body . the speed of the backward wave is higher than the forward speed of the fish . the waves of curvature on the bodies of undulatory swimmers are caused by waves of muscle activations running towards the tail with a 180 phase shift between the left and the right side ( videler and wardle 1991 ) . red muscles power the undulations during routine steady swimming up to a few body lengths per second , and the white muscles of the myotomes are mainly recruited during bursts of fast swimming and startle responses . fish can swim without getting exhausted at speeds up to approximately 2 body lengths per second while using red muscles only . at higher speeds , white muscles are used , and endurance decreases rapidly to less than a minute above 4 body lengths per second ( videler 1993 ) . their oxygen uptake is restricted because the gills are not functioning optimally due to the high viscosity conditions . that is the main reason why mortality of plaice larvae for example is 96% in the first month ; only 8 hatchlings out of 100,000 eggs survive the first year . green and fisher ( 2004 ) found that larvae of the fire clownfish ( amphiprion melanopus ) raised in temperatures of 25 instead of 28c had longer larval durations , reduced growth rates and were slower swimmers . growth curves of many species show an inflexion point indicating faster growth in length during the early stages ( fig . 3 ) ( mller and videler 1996 ) . larvae can escape the viscous regime , and a number of potential predators , by increasing in length and by swimming faster . muscles of fast growing carp larvae had a smaller cross - sectional area of muscles with more and thinner fibres than larvae growing slower on a different diet ( alami - durante et al . studies based on back calculations of otolith growth marks showed that faster growing individuals of bluefish ( pomatomus saltatrix ) larvae had a higher probability of survival ( hare and cowen 1997).fig . data from mcgurk 1985 the inflection point in the growth curve of a herring larva . data from mcgurk 1985 a link between condition and mortality was shown in a study of the bluehead wrasse ( thalassoma bifasciatum ) by grorud - colvert and ponaugle ( 2006 ) . the fastest swimmers among larval recruits caught immediately after settlement on the coral reef , grew faster and had a shorter vulnerable pelagic life phase . ( 2006 ) showed that selective mortality in the clupeid spratelloides gracilis removes small , slow - growing larvae . the size of larvae was principally determined by egg size . during the postlarval stages , growth curves of many fish species show fast increase in length during the juvenile phase . the brownface butterfly fish ( chaetodon larvatus ) , for example , grows very fast ( fig . 4 ) during the first year to reach adult size ( zekeria et al . 2006).fig . 4the growth curve of the brownface butterfly fish ( chaetodon larvatus ) in the southern red sea ( zekeria et al . 2006 ) the growth curve of the brownface butterfly fish ( chaetodon larvatus ) in the southern red sea ( zekeria et al . 2006 ) natural selection processes operate through increased predation on the smaller and least mobile individuals . small eggs , larvae and juveniles are consumed in large quantities by predators often including larger stages of the same species . in contrast to what happens under natural conditions , fish culture seeks to maximise yield during all stages from egg to adult . there is no special attention for the condition and growth of white muscles . compared with the situation in the wild , swimming opportunities are extremely restricted during all stages of development from larva to adult under most culture conditions . the variety of types of exercise is limited as well as the possibilities to practise certain skills , especially those involving white muscles . flow channels , induced rotational flows , and other training facilities for fish to improve fish quality in farms will not lead to increased white muscle mass because fish use predominantly red muscles to swim at these speeds ( see , i.e. , bjrnevik et al . on the other hand , forced swimming at moderate speeds of around 12 body lengths / s in adults has been shown to provide the best conversion of food into growth in some species ( see evidence reviewed in jobling et al . 1993 ; videler 1993 ) . it is unclear how much of the increase in body weight is due to white muscle mass enhancement . improved physiological performance and reduced frequencies of aggressive interactions are probably partly responsible for this effect . lack of exercise and high feeding rates make reared fish shorter and fatter ( blaxter and hunter 1982 ) . food usually comes in the form of pellets which do not require natural attack strategies . resulting stress will have a negative effect on healthy growth . crowding , even for schooling fish , seriously affects development and growth ( santos et al . 2010 ) . in atlantic salmon , crowding of more than 200 kg fish / m changes structural muscle proteins and enzymes involved in energy production ( veiseth - kent et al . it will cause unnatural and chaotic flow patterns and an overload of information through the lateral line system to the brain ( in humans information overload in the brain causes psychosis ) . fish farming also applies selection pressures on the farmed populations , either on purpose or as a side effect of management practises . these are aimed at maximum survival rates and will finally result in changes in the inherited properties of the species concerned . therefore , the quality of the white muscle can be expected to decrease as a consequence of the lack of selection for the use of that tissue throughout the development from egg to adult . regarding the fundamental differences between farming and natural conditions , it does not come as a surprise that the quality of the white muscle of farmed fish deviates from that of wild conspecifics ( santos et al . 2010 ) . the implicit goal in fish culture is the aim to produce fish for human consumption which can not be distinguished from wild fish . that goal could be approached by copying the natural selection processes as closely as possible during the entire life cycle of fish in captivity and by offering close to natural ecological conditions . the largest eggs should be selected to start the production process using larvae with the highest number of white muscle cells . during the egg stage , white muscle fibres develop depending on temperature and oxygen levels in a complicated and different way for each species . the general trend is that the biggest eggs produce the largest larvae with the highest survival chances . after hatching , hunting for live food will induce kick and glide swimming , which promotes white muscle development and growth . during the adult phase , animals should be able to hunt for or gather food in a natural way to maintain optimal conditions and to avoid the accumulation of fat inside the white muscle tissue .

due to rapid depletion of wild stocks , the necessity to cultivate fish is eminent . current fish farming practices seek to improve flesh quality . the notion that white muscles are the main target of the fishing industry is emphasized . a novel approach is suggested based on the development of white muscles in wild fish from eggs to adults . a compilation of facts about white muscle structure , function and ontogeny is followed by an account of the changes in swimming behaviour and performance related to the use of white muscle during growth from larva to adult . ecological data narrate early swimming performance with white muscle development and growth , unveiling some of the important natural selection factors eliminating weak swimmers and poor growers from the breeding stock . a comparison between fish culture practise and natural conditions reveals fundamental differences . new approaches following wild breeding processes promise several important advantages regarding the quality of white muscle .

Introduction Fish white muscles Swimming from larva to adult Ecology of larval swimming Fish farming practise in comparison with natural selection

PMC3015929

the field of minimally invasive surgery continues to advance rapidly , improving patient comfort , satisfaction , and outcomes . laparoscopy has become a proven and often preferred method to approach even the most complex operative problems . however , the need to remove resected specimens through an abdominal incision can at times limit the advantages of this modality , especially in regard to postoperative pain and recovery . this dilemma has , in part , prompted further development of techniques , including minilaparoscopy , natural orifice transluminal endoscopic surgery ( notes ) , and other less invasive methods of operating and extracting specimens from within the abdominal and pelvic cavities . the transvaginal approach for resection of the uterus has long been used by gynecologists for hysterectomy but of late has been modified by laparoscopic surgeons for the extraction of numerous organs including the gallbladder , appendix , kidney , bladder , and colon . moreover , both wilson et al and franklin et al have successfully performed completely laparoscopic right hemicolectomies utilizing intracorporeal anastomosis and transvaginal specimen extraction . nevertheless , the use of a working vaginal port during laparoscopy to reduce the number and size of abdominal incisions , as well as providing a means of large specimen extraction , has not been commonly described . tsin et al reported their 7-year experience using culdolaparoscopy with minilaparoscopy to perform various gynecologic operations , cholecystectomies , and appendectomies with success and few complications . more recently , lacy et al have applied a similar transvaginal approach to perform a radical resection of the sigmoid colon for adenocarcinoma . herein , we report the successful use of the laparoscopic - assisted transvaginal approach to sigmoidectomy and rectocolpopexy for treatment of rectal prolapse that highlights the feasibility and versatility of laparoscopic - assisted natural orifice surgery ( lanos ) . a 63-year - old woman presented with a 7-year history of worsening rectal prolapse associated with increasing pain as well as incontinence of flatus and stool . she had previously undergone total abdominal hysterectomy through a pfannenstiel incision but had no other history of abdominal or pelvic surgery . complete evaluation demonstrated significant , full - thickness rectal prolapse and an elongated sigmoid colon . the patient was offered a laparoscopic - assisted sigmoidectomy with rectocolpopexy through a novel transvaginal approach as described below . in the lithotomy position , a 5-mm trocar was introduced into the peritoneal cavity through the umbilicus and pneumoperitoneum was obtained . two additional 5-mm ports were placed transabdominally in the right and left lower quadrants to assist with mobilization of the colon and rectum . a 12-mm trocar was also placed transvaginally through the apex of the vagina and was used as a working port to retract the colon and introduce the laparoscopic stapler ( figure 1a ) . the sigmoid colon and rectum were fully mobilized from peritoneal attachments in a lateral to medial direction . the rectum was further mobilized along the endopelvic fascia to the pelvic floor , and anteriorly away from the vagina as distal as possible . the rectocolpopexy sutures ( 0 silk ) were brought into the pelvis through the vaginal port . they were placed bilaterally , first through the periosteum of the sacral promontory and then through lateral perirectal tissues and the uterosacral ligaments to have a tension - free fixation of the rectum and vagina to the sacrum . both ends of each suture a 5-mm , vessel sealing device ( ligasure , valleylab , boulder , co ) was used to divide the mesentery of the sigmoid and rectosigmoid colon , keeping the inferior mesenteric and superior rectal vessels intact . the rectosigmoid was transected by using a laparoscopic stapler ( endo gia , covidien , mansfield , ma ) placed through the vaginal port ( figure 1b ) . a surgical sponge was used to replace the vaginal trocar to retain pneumoperitoneum while extending the colpotomy , as well as to retain distention of the vagina and avoid thermal injury . the stapled end of the proximal colon was grasped with a long allis clamp , which was passed transvaginally , and the colon brought into the vagina . the vaginal introitus was then opened with a parks retractor and the redundant colon externalized ( figure 2 ) and transected . the proximal end of the colon was prepared for intracorporeal reanastomosis by introducing the anvil of a 28-mm , end - to - end anastomosis stapler ( eea , covidien , mansfield , ma ) and securing it with a 2 0 polypropylene , purse - string suture . the bowel was then returned into the abdomen and the colpotomy closed with running 2 0 polyglactin suture . after pneumoperitoneum was re - established , the eea stapler was introduced into the rectum and an intracorporeal anastomosis performed . the operation was then concluded following the evacuation of the co2 gas from within the peritoneal cavity and the removal of the laparoscopic trocars . a loose packing of gauze , impregnated with sulfanilamide cream , was placed into the vagina at the completion of the case and removed the following day . we successfully performed a laparoscopic - assisted sigmoidectomy and rectocolpopexy through a transvaginal approach to repair rectal prolapse in a 63-year - old woman . the patient tolerated the procedure well , complained of minimal postoperative pain , and did not require narcotics beyond the day of the operation . she was started on a diet the morning after the operation and was discharged from the hospital on postoperative day 3 following the return of bowel function . at 3-month follow - up , she remained without prolapse and had had no complications . the use of a transvaginal approach to lanos is now within the reach of the well - trained laparoscopic surgeon . in our experience , total operating time is equivalent to traditional laparoscopic - assisted surgeries , because closure of the colpotomy requires no more time than closure of an abdominal incision . however , from a technical stand point , it should be noted that the actions performed through the transvaginal port are often mirror imaged when one is working in the pelvis . nonetheless , we believe that the use of a working transvaginal port only serves to enhance an operator 's ability and have not found it to be awkward . the largest experience of laparoscopic colorectal resections with transvaginal specimen extraction to date has been published by ghezzi et al and boni et al . they describe their use of this technique in the treatment of endometriosis and indicate that extraction of specimens through the vagina appears to be safe and without increased risk of major complications , such as pelvic infection or vaginal fistula . complications necessitating reoperation , as may occur with anastomotic leak , should be approached in the same manner as following any traditional laparoscopic surgery . in the emerging era of natural orifice surgery , patients are willing to accept novel surgical techniques if they provide superior outcomes and reduce pain and scaring . the use of a working transvaginal port in lanos offers a unique solution for applying minimally invasive techniques to operations that require larger instruments and abdominal incisions for specimen removal . as lanos continues to evolve , the laparoscopic - assisted transvaginal approach to abdominal and pelvic surgery should be further examined as a practical option for the improvement of patient care and satisfaction . to this end , we are currently performing and evaluating laparoscopic - assisted transvaginal colonic resections for various indications in a prospective manner and will continue to report our results .

background and objectives : laparoscopic - assisted colonic resection has been well described for multiple surgical indications . this typically requires an abdominal incision for specimen removal that is associated with the majority of postoperative pain . we describe the first laparoscopic - assisted transvaginal approach for sigmoidectomy and rectocolpopexy for the treatment of rectal prolapse.methods:mobilization of the sigmoid colon was performed laparoscopically using a 12-mm vaginal port and 3 additional 5-mm abdominal ports . a laparoscopic stapler was used through the vaginal port to transect the distal sigmoid colon . the specimen was subsequently externalized through the colpotomy to complete the resection and prepare the remaining bowel for intracorporeal , end - toend , stapled anastomosis . the colpotomy was then repaired , and the colorectal anastomosis and rectocolpopexy were completed laparoscopically.results:sigmoidectomy and rectocolpopexy were successfully performed laparoscopically by using a transvaginal approach without the need for an abdominal incision for specimen removal . the patient had an uncomplicated postoperative course , complained of minimal pain , and was discharged home on postoperative day 3.conclusions:laparoscopic-assisted transvaginal sigmoidectomy and rectocolpopexy is a feasible option that appears to be associated with little incisional pain and rapid recovery .

INTRODUCTION METHODS RESULTS DISCUSSION CONCLUSION

PMC4327500

an optimal adaptation of the denture bases to its supporting structures is closely related to the retention of the denture and health and preservation of the underlying tissues . however , residual ridge reduces over an indefinite period of time and these gradual changes of oral tissues require that complete or partial dentures be relined to improve their adaptation to the supporting tissues7,15 . autopolymerizing reline resins offer an immediate and relatively simple means to directly recondition the denture base surface of ill - fitting prostheses7 . despite these advantages , several autopolymerizing reline resins present lower flexural strength than heat - polymerized acrylic resins2,23,29 and the flexural strength of heat - polymerized acrylic resins can be significantly decreased after relining2,21,23 . this is due to the fact that there is higher residual monomer in autopolymerizing acrylic resins than in heat - polymerized acrylic resins13,27,28 . the reason for the higher residual monomer content in the autopolymerizing acrylic resin is the low degree of conversion achieved by the use of a chemical activator as opposed to that generated by heat activation26 . as a result this residual monomer acts as a plasticizer , thus resulting in deleterious effect on the mechanical properties of denture base resins5,13 . in addition , residual monomer resulting from incomplete conversion of monomers into polymer has the potential to elicit irritation , inflammation and an allergic response by oral mucosa4 . in order to overcome these limitations , several techniques to increase the degree of conversion of autopolymerizing acrylic resins and thus reduce the residual monomer content have been advocated , such as immersion in hot water11;16,24,25 and microwave irradiation5,16,25,29,30 . however , there are no reports in the literature comparing the influence of these two postpolymerization treatments on the physicomechanical properties of acrylic resins . the aim of this study was to determine the effect of waterbath and microwave post - polymerization treatments on the flexural strength and vickers hardness of four different autopolymerizing reline resins and one heat - polymerized acrylic resin . the hypothesis tested was that the flexural strength and vickers hardness of the reline and acrylic denture base resins would be improved by the post - polymerization treatments . four autopolymerizing reline resins were selected for comparison to a conventional heat - polymerized acrylic resin ( table 1 ) . tokuso rebase fast and ufi gel hard c contain high percentages of cross - linking agent . the liquid composition of kooliner is isobutyl methacrylate , without a cross - linking agent , whereas duraliner ii liquid contains butyl methacrylate and a cross - linking agent . lucitone 550 heat - polymerized acrylic resin was selected as representative of the poly(methyl methacrylate ) heat - polymerized acrylic resins , which are commonly used for the fabrication of denture bases . pema , poly ( ethyl methacrylate ) ; pmma , poly ( methyl methacrylate ) ; bma , butyl methacrylate ; ibma , isobutyl methacrylate ; 1,6 - hdma , ( 1,6-hexanediol dimethacrylate ) ; maop , -methacryloyl oxyethyl propionate ; mma , methyl methacrylate ; edgma , ( ethylene glycol dimethacrylate ) each material was mixed according to the manufacturer 's instructions and applied to a stainless steel mold ( 64 10 3.3 mm ) placed on an acetate sheet and a glass slab . a second acetate sheet and glass slab was placed over the material and light pressure was applied to expel excess material from the mold . for ufi gel the sides of all specimens were ground with 400-grit silicon carbide paper ( 3 m , st . the accuracy of the dimensions ( width and thickness ) was verified with a micrometer ( mitutoyo sul americana , suzano , sp , brazil ) , accurate to 0.01 mm , at 3 locations of each dimension to within 0.02 mm tolerance8 . the final specimen dimensions were 64 mm length , 10 0.02 mm width and 3.3 0.02 mm height8 . to fabricate lucitone 550 specimens , initially , silicone impression material ( optosyl confort , heraeus kulzer , dormagen , germany ) was adapted inside the stainless steel mold . the silicone patterns were invested by sandwiching them between 2 glass slides in type iv stone ( vel - mix , kerr , romulus , mi , usa ) , using a conventional denture processing flask ( bethil ind e co ltda , marlia , sp , brazil ) . lucitone 550 material was mixed , packed under pressure and processed according to the manufacturer 's recommendations ( table 1 ) . considering that the manufacturer of lucitone 550 recommends two different polymerization cycles , two groups ( n=30 ) of denture base resin specimens were prepared and polymerized using either the short ( 90 minutes at 73c and 100c for 30 minutes ) or the long cycle ( 9 hours at 71c ) . after polymerization , the flasks were bench cooled at room temperature for 30 minutes and for 15 minutes under running water before the specimens were removed from the flasks . all specimens were finished as described , had the dimensions verified and were stored in water at 37 1c for 48 2 hours before testing8 . the control group of each material remained as finished . for water - bath group , the specimens were submitted to a postpolymerization treatment in a water - bath at 55c . the autopolymerizing reline resin specimens were held at this temperature for 10 minutes , as suggested by the manufacturer of duraliner ii to reduce the residual monomer taste . lucitone 550 specimens were held at this temperature for 60 minutes , following the recommendation for denture base resins reported by tsuchiya , et al.24 the specimens were then bench cooled to room temperature before testing . microwaved specimens received post - polymerization treatment by microwave irradiation in a domestic adjustable - wattage microwave oven with a turntable ( bmc38abhna , brastemp da amazonia s.a . , manaus , brazil ) and irradiating with : 550 w/3 min lucitone 550 ; 650 w/4 min duraliner ii ; 550 w/5 min kooliner ; 500 w/5 min tokuso rebase fast and 550 w/4 min ufi gel hard c. power / time setting for each material were determined in a preliminary study , which evaluated the effect of 9 different power / exposure time combinations on the flexural strength of the materials investigated in this study29 . the power / time setting , which produced the highest flexural strength value for each material , was used in the present study . all specimens were subjected to flexural strength testing in a servo - hydraulic universal testing machine ( mts 810 , mts systems corporation , eden prairie , mn , usa ) using 3-point loading . a crosshead speed of 5 mm / min was used and the distance between the supports was 50 mm . load was applied until failure and fracture load was recorded in n. flexural strength ( mpa ) was calculated using the equation : fs = 3 wl/2 bd , where fs is the flexural strength , w is the maximum load before fracture ( n ) , l is the distance between the supports ( 50 mm ) , b is the width of the specimen ( mm ) , and d is the thickness of the specimen ( mm)8 . after flexural strength test , one fragment of each specimen was selected for microhardness test . vickers indentation ( vhn ) is a valid tool for evaluating the hardness and viscoelastic responses of polymers14 , and some studies have used the vhn test to evaluate the hardness of denture base acrylic resin17 and acrylic resin denture teeth18 . measurements of vhn were made with a microhardness indenter machine ( micromet 2100 ; buehler , lake bluff , il , usa ) at a 25 gf load for 30 seconds . when duraliner was tested , the load was 10 gf , so that the indentation could be properly measured . the operator of the test machine read the lengths of the diagonals immediately after each indentation , with a minimal ( as short as 10 seconds ) period of time elapsed between making and reading the indentations . it was assumed that due to the short time elapsed between making and reading the indentation , the viscoelastic recovery of the diagonals after indentation was minimal14 . the operator measured the diagonals , and the equipment automatically converted these measurements to vhn numbers ( kg / mm ) with a scale of 1 digit to the right of the decimal point in a number . the influence of the main factors ( material and treatment ) on the flexural strength and vickers hardness was analyzed by two - way analysis of variance ( anova ) , followed by tukey 's test ( =0.05 ) . all specimens were subjected to flexural strength testing in a servo - hydraulic universal testing machine ( mts 810 , mts systems corporation , eden prairie , mn , usa ) using 3-point loading . a crosshead speed of 5 mm / min was used and the distance between the supports was 50 mm . load was applied until failure and fracture load was recorded in n. flexural strength ( mpa ) was calculated using the equation : fs = 3 wl/2 bd , where fs is the flexural strength , w is the maximum load before fracture ( n ) , l is the distance between the supports ( 50 mm ) , b is the width of the specimen ( mm ) , and d is the thickness of the specimen ( mm)8 . after flexural strength test , one fragment of each specimen was selected for microhardness test . vickers indentation ( vhn ) is a valid tool for evaluating the hardness and viscoelastic responses of polymers14 , and some studies have used the vhn test to evaluate the hardness of denture base acrylic resin17 and acrylic resin denture teeth18 . measurements of vhn were made with a microhardness indenter machine ( micromet 2100 ; buehler , lake bluff , il , usa ) at a 25 gf load for 30 seconds . when duraliner was tested , the load was 10 gf , so that the indentation could be properly measured . the operator of the test machine read the lengths of the diagonals immediately after each indentation , with a minimal ( as short as 10 seconds ) period of time elapsed between making and reading the indentations . it was assumed that due to the short time elapsed between making and reading the indentation , the viscoelastic recovery of the diagonals after indentation was minimal14 . the operator measured the diagonals , and the equipment automatically converted these measurements to vhn numbers ( kg / mm ) with a scale of 1 digit to the right of the decimal point in a number . the influence of the main factors ( material and treatment ) on the flexural strength and vickers hardness was analyzed by two - way analysis of variance ( anova ) , followed by tukey 's test ( =0.05 ) . two - way anova for the flexural strength results showed that significant differences were found for the 2 main factors ( material and treatment ) and their interaction ( p<0.001 ) . table 2 shows that lucitone 550 ( short cycle ) microwaved specimens exhibited significantly higher flexural strength than its respective control group . water - bath post - polymerization treatment promoted a significant increase in the flexural strength of kooliner and lucitone 550 ( long cycle ) . the flexural strength of the reline materials duraliner ii , ufi gel hard c and tokuso rebase fast were not affected by any of the postpolymerization treatments . within each line , entries with the same uppercase letter were not significantly different ( p=0.05 ) . the same superscript letter in a column represents values that were not significantly different at p = 0.05 . when the flexural strength of the materials were compared , lucitone 550 ( short cycle ) exhibited significantly higher flexural strength than ufi gel hard c and lucitone 550 ( long cycle ) in both control and microwave groups . in these groups , no significant differences were found between the heat - polymerized acrylic resin lucitone 550 ( short cycle ) and the autopolymerizing reline resin tokuso rebase fast , which , in turn , was not significantly different from ufi gel hard c and lucitone 550 ( long cycle ) . no significant difference was found between kooliner and duraliner ii controls , whereas kooliner showed significantly higher flexural strength than duraliner ii after both water - bath and microwave post - polymerization treatments . for the specimens submitted to water - bath post - polymerization treatment , there were no significant differences among lucitone 550 ( short and long cycles ) , ufi gel hard c , and tokuso rebase fast materials . in addition , no significant difference was observed between kooliner and lucitone 550 ( short cycle ) . two - way anova for vickers hardness results indicated that significant differences were found only for the factor material ( p<0.001 ) . table 3 presents the hardness means of the materials and the results of tukey 's hsd pos hoc test ( =0.05 ) . no significant difference was observed between lucitone 550 specimens polymerized using either short or long cycle . tokuso rebase fast material showed lower vhn values than lucitone 550 and higher than kooliner and duraliner ii resins , which were not significantly different from each other . the same uppercase letter in a column represents values that were not significantly different at p = 0.05 . based on the present methodology , the hypothesis that the flexural strength of autopolymerizing and heat - polymerized acrylic resins could be affected by the post - polymerization treatments was accepted . the results demonstrated that water - bath post - polymerization treatment increased the flexural strength of kooliner and lucitone 550 ( long cycle ) . first , the residual monomer content of specimens submitted to water - bath postpolymerization treatment might have been reduced , in part , by diffusion of the monomer molecules into the water27 . second , the existence of free radicals in the polymer chains would favor the occurrence of the continuous polymerization phenomenon11 . these hypotheses are supported by the results of an earlier report25 , in which immersion in hot water promoted a significant reduction in the residual monomer content for all materials evaluated in the present investigation . for microwave post - polymerization treatment , the flexural strength of lucitone 550 specimens ( short cycle ) a plausible explanation for this result is the further polymerization produced by microwave irradiation , thereby reducing the residual monomer content1,5 . in this study , lucitone 550 material was polymerized according to the manufacturer 's instructions , which included a terminal boil for 30 minutes . previous studies have shown that heat - polymerized acrylic resins should be maintained for at least 1 hour in terminal boiling to achieve the maximum monomer conversion6,28 . probably , the short boiling time adopted in this study resulted in lower degree of conversion , which might have been improved after microwave post - polymerization treatments . the favorable effect of microwave post - polymerization treatment was also observed in the studies by yunus , et al.30 and polyzois , et al.19 , who reported that the strength of specimens of denture base resins repaired with autopolymerizing acrylic resins was improved by effective microwave irradiation . similarly , blagojevic and murphy5 showed that microwaving of a polymerized autopolymerizing reline resin for 3 minutes at 600 w improved its mechanical properties and reduced residual monomer by almost a factor of four . the flexural strength of ufi gel hard c , tokuso rebase fast and duraliner ii was not significantly improved by any of the post - polymerization treatments . the results obtained for tokuso rebase fast and ufi gel hard could be attributed to their composition . tokuso rebase fast and ufi gel hard c are composed of poly ( ethylmethacrylate ) and their liquid contains a cross - linking agent , 1,6-hexanediol dimethacrylate ( 1,6-hdma)2,3 . the long distance between the 2 methacrylate groups of 1,6-hexanediol dimethacrylate results in more reactivity of the second methacrylate group22,29 . therefore , a high degree of conversion of dimethacrylates contained in ufi gel hard c and tokuso rebase fast may occur by the chemical activation reaction , thereby resulting in a highly cross - linked polymer . for duraliner ii , one factor that may have influenced its behavior is the lowest powder / liquid ratio10 . according to lamb12 , with the lower powder / liquid ratio , the ratio of peroxide / amine will be lowered and hence the rate of decomposition of the peroxide increased . consequently , the net effect will be a decrease in the concentration of longer - lived radicals , which are essential for the post - polymerization reaction . comparing the control groups , lucitone 550 specimens processed using long cycle polymerization , which did not include a period at 100c , produced specimens with the lower flexural strength values than lucitone 550 specimens using short cycle . these results may be attributed to the variation of time and temperature during polymerization , which can influence the amount of residual monomer content in the polymerized acrylic resin6,28 . a previous study25 has demonstrated that the terminal boiling ( 100c ) period included in the short polymerization cycle produced specimens with lower level of residual monomer than those polymerized by the long cycle . this may explain the higher flexural strength of the specimens of lucitone 550 , polymerized using the short cycle9 . favorably , the flexural strength values of the autopolymerizing reline resin tokuso rebase fast were not significantly different from that of the heat - polymerized acrylic resin lucitone 550 ( long cycle and short cycle ) . likewise , ufi gel hard c did not differ from lucitone 550 polymerized using the long cycle . these findings are probably related to the fact that these reline materials are both highly cross - linked polymers3 and support the observations from clinical trials in which tokuso rebase fast demonstrated excellent clinical performance after 12-month observation7,15 . on the other hand , although duraliner ii is also a cross - linked material , it produced a low flexural strength mean . as previously stated , the low powder / liquid ratio used for duraliner ii may have accounted for this result . from the clinical standpoint , the results indicated that kooliner and duraliner ii should be accepted as temporary materials due to the limited mechanical properties presented . however , as the flexural strength obtained with kooliner was comparable to that of lucitone 550 ( short cycle ) after water - bath post - polymerization treatment , this procedure could be used to improve the longevity of the denture bases relined with this material . the hypothesis that the vickers hardness of autopolymerizing and heat - polymerized acrylic resins tested would be influenced by post - polymerization treatments was rejected . similar finding was found by blagojevic and murphy5 , who demonstrated that autopolymerizing acrylic resin had no difference in hardness values before and after microwaving at 600 w for 3 minutes . these results , taken together with those from flexural tests , suggest that the effect of the postpolymerization treatments was more pronounced in the bulk of the specimens rather than in their superficial layer . this theory is supported by other study16 , in which higher hardness values were recorded at greater specimen depths after an additional cycle of polymerization using either microwave energy or hot water . the absence of any detectable effect of water - bath and microwave irradiation treatments on the superficial layer of the tested materials may also be explained by the presence of oxygen , which may have competed for free radicals during the post - polymerization treatment . the reactivity of oxygen with free radicals has been reported to be higher than that of free radicals with monomers13 . although tokuso rebase fast and ufi gel hard c contain the same cross - linking agent in the liquid , it was interesting to note that the former showed significantly lower hardness values . this may be explained by the use of the monofunctional monomer , 39.8% -methacryloyl oxyethyl propionate ( maop ) that constitutes the liquid component of tokuso rebase fast material . the flexibility of the polymer chain may account for the relatively lower hardness of tokuso rebase fast in comparison to ufi gel hard c. it could be also hypothesized that the greater surface hardness of ufi gel hard c was due to the inclusion of inorganic filler particles and the highly cross - linked polymer structure . to the best of the authors ' knowledge , no other studies have evaluated extensively this new material since its introduction to the market . studies investigating ufi gel hard c composition must be gathered to validate the current hypothesis . as expected , lucitone 550 specimens polymerized by short and long cycles were significantly harder than those made with tokuso rebase fast , kooliner and duraliner ii . finally , it was not surprising that kooliner and duraliner ii showed the lowest vickers hardness values of all materials . according to rawls20 , the butyl / isobutyl methacrylate molecules increase the backbone separation of the polymer molecules , decreasing the intermolecular interaction . thus , the isobutyl ( kooliner ) possesses a lower surface hardness , and the n - butyl ( duraliner ii ) has an even lower hardness . the lowest hardness values observed for duraliner ii could also be related to its low powder / liquid ratio , which will result in a high residual monomer left in the polymerized resin10,25 . based upon the limitations imposed in the present study , these findings suggest that post - polymerization treatments offer advantages on the flexural strength of some tested materials . however , the interpretation of the results from this report must be made with caution and further investigations are required in order to clarify any clinical influence of the water - bath and microwave post - polymerization treatments on the longevity of intact and relined denture bases . under the experimental conditions used in this study the following conclusions can be drawn : the flexural strength of the heat - polymerized resin lucitone 550 ( short cycle ) was increased after microwave postpolymerization treatment.after water bath post - polymerization treatment , the heat - polymerized resin lucitone 550 ( long cycle ) and autopolymerizing resin kooliner showed an increase in flexural strength values.duraliner ii and kooliner showed the lowest flexural strength among the relining materials . the autopolymerizing resins tokuso rebase fast and ufi gel hard c exhibited flexural strength comparable to that of the conventional heat - polymerized lucitone 550 , regardless of the polymerization cycle.no significant effect of post - polymerization treatments was observed on the vickers hardness.ufi gel hard c demonstrated significantly higher vickers hardness compared to the other tested materials . vickers hardness of lucitone 550 was significantly higher than that of tokuso rebase fast , duraliner ii and kooliner . the flexural strength of the heat - polymerized resin lucitone 550 ( short cycle ) was increased after microwave postpolymerization treatment . after water bath post - polymerization treatment , the heat - polymerized resin lucitone 550 ( long cycle ) and autopolymerizing resin kooliner showed an increase in flexural strength values . the autopolymerizing resins tokuso rebase fast and ufi gel hard c exhibited flexural strength comparable to that of the conventional heat - polymerized lucitone 550 , regardless of the polymerization cycle . ufi gel hard c demonstrated significantly higher vickers hardness compared to the other tested materials . vickers hardness of lucitone 550 was significantly higher than that of tokuso rebase fast , duraliner ii and kooliner .

this study evaluated the effect of water - bath and microwave post - polymerization treatments on the flexural strength and vickers hardness of four autopolymerizing reline resins ( duraliner ii - d , kooliner - k , tokuso rebase fast - tr and ufi gel hard c - ugh ) and one heat - polymerized acrylic resin ( lucitone 550-l ) , processed using two polymerization cycles ( short cycle - 90 minutes at 73c and 100c for 30 minutes ; and long cycle - 9 hours at 71c ) . for each material , thirty specimens ( 64 x 10 x 3.3 mm ) were made and divided into 3 groups ( n=10 ) . specimens were tested after : processing ( control group ) ; water - bath at 55c for 10 minutes ( reline materials ) or 60 minutes ( l ) ; and microwave irradiation . flexural strength tests were performed at a crosshead speed of 5 mm / min using a three - point bending device with a span of 50 mm . the flexural strengths values were calculated in mpa . one fragment of each specimen was submitted to vickers hardness test . data were analyzed by 2-way anova followed by tukey 's hsd test ( =0.05 ) . l microwaved specimens ( short cycle ) exhibited significantly higher flexural strength means than its respective control group ( p<0.05 ) . water - bath promoted a significant increase ( p<0.05 ) in flexural strength of k and l ( long cycle ) . the hardness of the tested materials was not influenced by the post - polymerization treatments . post - polymerization treatments could be used to improve the flexural strength of some materials tested .

INTRODUCTION MATERIAL AND METHODS Flexural Strength Test Microhardness Test RESULTS DISCUSSION CONCLUSIONS

PMC3181776

the duration of the peak of melatonin secretion is positively correlated with the length of the night period . experimentally , it has been demonstrated that the brain is able to integrate photoperiodic information through these changes in duration of melatonin synthesis . this explains the current use of this hormone in farming to control seasonal functions ( eg , fur growth , reproduction , and milk production ) . this also opens therapeutic perspectives if we consider the hypothesis of wehr that the photoperiod - induccd changes in the duration of melatonin secretion drive the annual cycle that occurs in seasonal affective disorders . the duration of nocturnal melatonin production is the key signal , but the existence within this signal of a melatonin - driven circadian rhythm of sensitivity to melatonin has been proposed to explain the photoperiodic response . in fact , our understanding of melatonin 's physiological functions depends on the understanding of how and where its action is exerted . considering the lipophilic nature of the hormone , interactions with specific intracellular proteins or nuclear receptors can not be excluded ; however , melatonin seems to exert its effects principally throughout high - affinity g - protein - coupled receptors . the introduction of 2-[-i]iodomelatonin ( i - mel ) heralded the development of the melatonin receptor field . the cloning of the first high - affinity melatonin receptor in 1994 by rbisawa et al then led to the subsequent identification of three types of vertebrate melatonin receptors ( mt1,mt2 , and mellc ) , and this very probably is only the beginning of a long list . considering the photoperiodic responses , the melatonin receptors involved most probably are of the mt1 subtype . indeed , the gene of the only other melatonin receptor subtype found in mammals , mt2 , is nonfunctional in two highly photoperiodic species , siberian and syrian hamsters ( weaver and reppert , unpublished data cited in reference 20 ) . the target sites mediating melatonin control of photopcriod - dependent seasonal functions and especially the annual sexual cycle have not yet been totally determined . contrary to what is generally claimed , melatonin receptors are present in a large number of structures in mammals ( more than 110 brain structures have been identified , among them the internal granular layer and the external plexiform layer of the olfactory bulb , lateral septum , septohippocampal nucleus , caudate putamen , bed nucleus of the stria terminalis , scn , mediobasal hypothalamic nuclei , paraventricular nuclei of the hypothalamus , paraventricular nuclei of the thalamus , intergeniculate leaflet , central and medial amygdaloid nucleus , inferior colliculus , fasciculus retroflexus , substantia nigra , and frontal , orbitofrontai and parietal cortex ; numerous peripheral organs also contain melatonin receptors 25 ) . however , a great variability has been noted in the number and location of structures among the species , as well as large differences in receptor density between structures and in the same structures between species . few structures are common , even among species from the same family , and very probably this should be correlated to either the numerous photoperiodic responses , which are different from one species to another , or the many different effects described for melatonin . one structure , however , the pars tuberalis ( pt ) of the pituitary , which contains a very high density of melatonin receptors in all mammals studied , is thought to be of primary importance in photoperiodic response . its density of melatonin receptors exhibits clear seasonal changes in photoperiodic species , but not in nonphotoperiodic mammals , and its implication in the control of seasonal secretion of prolactin has been demonstrated . the pt is thus a good model to delineate the melatonin 's signal transduction pathways and to study how the cellular response can distinguish between long- and short - duration melatonin signals . the cyclic adenosine monophosphate ( camp)-mediated pathways appear to be central to the melatonin readout . pretreatment with melatonin has been demonstrated to induce a sensitization of adenylate cyclase , and a potentiated camp response to forskolin stimulation . melatonin pretreatment that is effective in potentiating camp accumulation in the pt is duration - dependent ( between 0 - 16 h ) and corresponds well with the duration of the nocturnal melatonin signal . the nocturnal melatonin signal is also crucial for the rhythmic expression in the pt of several camp - responsive genes , including the transcriptional inhibitor - inducible camp early repressor ( icrr ) , and of several clock genes . indeed , two components of the molecular clock , namely perl and cry 1 , are rhythmically expressed in the ft furthermore , other components of the clock like timeless , clock , and perl ( pvet p et al , unpublished observations ) are also expressed in the pt , at least in the pt of some rodents , raising the possibility that the pt might contain a complete set of clock genes . however , the clock gene expression in the pt differs from what is observed in the scn or other peripheral tissues ( peripheral oscillators ) because it appears to be directly driven by melatonin . removal of the pineal gland abolishes rhythmic pt gene expression , and extension of the dark phase of the ld cycle dampens the amplitude of the perl in pt cells . cry1 is rapidly and very strongly induced by melatonin administration . in nontrcated animals , a peak of expression occurs during the dark phase ( ie , at a time when melatonin is present in the bloodstream ) . this indicates that melatonin may gate the expression of cry1 in the pt , suggesting that these clock genes are involved in the melatonin readout mechanism . it acts as a sensor of melatonin onset , rather than a marker of the duration of the melatonin signal . per1 mrna peaks early in the day , when blood plasma melatonin levels are back to low levels . this dual effect of melatonin together with its photoperiod - dependent pattern in plasma levels may provide the basis of a time measurement mechanism . this model may help understand how the pt is involved in the seasonal control of prolactin secretion by the pt . the validation of such a model will , however , require further experiments and the complete understanding of the melatonin and photoperiodic readout requires a link with identified downstream response in the pr this is still difficult . it is through the production of a prolactin - releasing ( or release inhibitor ) factor that the photoperiodic and melatonin information to lactotroph cells in the pituitary arc relayed . photoperiod - induced changes in prolactin secretion , however , are not enough to explain the seasonal sexual cycle . this implies that in order to mediate photoperiodic information melatonin must act on other target sites . this view is supported by the fact that syrian hamsters bearing lesions to the dorsomedial hypothalamus ( dmh ) and infused with melatonin to mimic short photoperiod ( sp ) , display differential responses in terms of prolactin and luteinizing hormone ( lh ) . while the prolactin response remains intact , the lh response in blocked by the dmh lesion . moreover , in sheep , melatonin implants in the mediobasal hypothalamus block the effects of sp on lh but not on prolactin , while implants close to the pt inhibit prolactin secretion . interestingly , melatonin binding sites have been detected in the dmh in the syrian hamster , although with a very low density , and their density depends on the photoperiod ( pvet p et al , unpublished data ) . the hypothesis of a parallel and concomitant action of melatonin on different structures to transduce the photoperiodic message is attractive . the photoperiod is known , through changes in duration of melatonin secretion , to control not only the reproductive annual cycle , but also a large number of other seasonal functions ( eg , body weight , hibernation , daily torpor , fur color changes , and migration ) . furthermore , not all seasonal functions are expressed in every species and different control mechanisms may be involved . for example , sp induces an activation of the sexual axis in sheep , but inhibition in syrian and siberian hamsters ; and hibernation in the syrian hamster is directly dependent on photoperiod , while in the ruropean hamster it is dependent on a circannual clock it thus seems likely that melatonin acts at different structures according to the species and the function . this concept would account for the large interspecies differences observed in mammals in the distribution of structures containing melatonin receptors . interestingly , and in support of this concept , a pharmacological dissociation of photopcriodic - controlled seasonal functions has been reported . s 22153 , a melatonin antagonist of mt1 and mt2 melatonin receptor subtypes , caused a decrease in the duration of hibernation in syrian hamsters under sp and low temperature , but did not affect sp - induced gonadal atrophy . in most nonmammalian vertebrates , the rhythmic synthesis and secretion of melatonin is the direct output of circadian clock , and the rhythmic changes in the concentration of circulating melatonin are fundamental to circadian rhythmicity . in mammals , despite the presence of melatonin receptors in the scn of most species indicating hormonal feedback on the clock , the consensus has been that melatonin has only a limited role in circadian organization . this view has arisen , in part , since pinealectomy has little effect on circadian organization . melatonin rhythm , however , is only one of the outputs of the clock and it is probable that , for the organization of circadian activities , a number of different output signals from the clock are involved in the distribution of circadian information to target tissues . after pinealectomy , for example , subtle desynchrony of several physiological functions has been described , and the reentrainment of the rat locomotor activity rhythm is modified after a phase shift of the ld cycle . one week after pinealectomy , the firing rate rhythm of scn neurons in vitro is altered , as well as the daily rhythm of responsiveness to melatonin . it is also known that melatonin interferes with metabolic activity ( glucose utilization and protein synthesis ) in the scn . the scn may use the daily melatonin signal to convey the circadian message to any system that can read it , ie , to any structure or organ possessing melatonin receptors , either in the central nervous system or at the periphery . this concept helps explain numerous results in the literature : the melatonin inhibition of spontaneous and light evoked activity of cells in the intergeniculate leaflet ; melatonin - enhancing splenic lymphocyte proliferation ; melatonin - induced inhibition of leukocyte rolling and adhesion to rat microcirculation58 ; melatonin - induced vasoconstriction of cerebral and tail arteries ; and melatonin regulation of emotional behavior . what could be the mechanism involved ? it appears that cyclical expression of these genes in the periphery is driven by the scn . the role of melatonin in regulating rhythmic clock gene expression in peripheral tissues as described in the pt ( see above ) may be one of the mechanisms for tissue - specific regulation of the phase of rhythmicity . interestingly , it has been demonstrated that the circadian rhythm of melatonin receptor density in rat pt is suppressed after pinealectomy and melatonin drives this rhythm directly . rven if the role of endogenous melatonin on clock functioning is not yet defined , the presence of melatonin receptors within the scn indicates that exogenous melatonin affects circadian regulation , which is of potential therapeutic value . exogenous melatonin is known to be able to influence , directly or indirectly , the phase and/or the period of the circadian clock . in terms of treatments , this means that exogenous melatonin ( or any agonist ) can be used as a pharmacological tool to manipulate sleep - wake cycle and other circadian rhythms ( chronobiotic properties ) . it has long been known that administration of melatonin can entrain free - running activity rhythms in rodents . entrainment means that the period of the observed rhythm must adjust to , and equal , the synchronizer ( zeitgeber ) cycle ( t ) , and a stable phase relation must be established between the rhythm and the zeitgeber cycle . this synchronization process occurs through daily phase shifts . administering melatonin for a series of t values , 24 h , 23 h 50 min , 23 h 45 min , 23 h 35 min , and 23 h 25 min has led to the definition of the limiting phase advance value to which the rat activity rhythm entrains to melatonin at 35 min . the entrainment limits found in this study correspond quite well to the maximum daily phase shift values defined by the melatonin phase response curve , and the magnitude of phase shift responses to a single melatonin injection ( range 15 - 52 min ) . daily acute melatonin administration in the rat thus causes true entrainment as defined by rnright et al . interestingly , when melatonin is administered by daily infusion , the phase angle difference between the entrained rhythm and the zeitgeber ( melatonin ) depends upon the duration of the infusion period . a negative phase angle is observed and its value increases with the duration of the infusion period . moreover , with long infusion times ( 8 h and , more especially , with 16 h ) , melatonin induces a change in the free - running period in the first days , suggesting that melatonin delays the pacemaker each day until entrainment occurs . in other words , with a long duration of infusion , entrainment occurs earlier than predicted by the model based on acute melatonin administration . these observations suggest that , beside its chronobiotic properties , melatonin affects the circadian clock properties ( effect on the period t ? ) . melatonin induced entrainment after a time during which circadian periods were either lengthened ( in a fraction of the animals ) or shortened ( in the others ) . this finding suggests that , to achieve entrainment , melatonin has to induce either a phase delay ( when the period is shortened ) or a phase advance ( when the period is lengthened ) . for example , when rats received a 5 h phase advance of the dark onset in ld conditions , those injected melatonin daily at the new dark onset reentrained with decreased latency ; some of the animals did so by phase delays , whereas others did so by phase advances . melatonin has been reported to entrain hamsters and arvicanthis ansorgei , a diurnal rodent , by inducing phase advances when the free - running period is longer than 24 h and phase delays when the period is shorter than 24 h. all these observations strongly suggest that the effects of exogenous melatonin are complex and depend on the period before entrainment . another potential effect of exogenous melatonin should be considered . a single application of melatonin within the scn , in vivo , induced a long - lasting increase in the amplitude of the nocturnal melatonin secretion . this effect demonstrates that exogenous melatonin is able to sustain the oscillation of the clock and suggests a possible role for endogenous melatonin in mammals . in the experiments reported above , responsiveness to melatonin is restricted to a narrow window of sensitivity , which is generally late in the subjective afternoon , but depends upon the duration of the melatonin signal as well as the previous free - running period . the finding that pinealectomized rats entrain to daily melatonin administration indicates that endogenous melatonin is not necessary for the entrainment effect of exogenous melatonin , for example , by entraining a window of sensitivity to melatonin . consequently , the effects of melatonin administration in vivo appear not to be related to the role of endogenous melatonin on circadian function . this conclusion is reinforced by the observation that to obtain entrainment of the circadian activity rhythm of rodents kept under constant darkness ( dd ) , high doses of melatonin have to be used , independently of the mode of administration . ' ill esc doses of melatonin produce peak serum levels 100- to 1000-fold higher than the endogenous melatonin nighttime levels . the necessity of such a high dose of melatonin is unlikely to be a consequence of its rapid metabolism . appropriate photoperiodic response is indeed obtained when melatonin is administered via a similar subcutaneous infusion system with a dose that mimics the endogenous secretion profile . most likely , this high dose of melatonin is needed because it is an integral part of the response observed . in vitro administration of melatonin can phase shift the firing rate of scn neuron brain slices ( rat and mouse ) . it is principally for this reason that it is generally believed that melatonin mediates these effects through the highaffinity melatonin receptors located within the scn . the high correlation between the density of melatonin receptors within the scn and the ability of daily melatonin administration to entrain the free - running activity rhythm in mammals supports this view . in contrast to the rat , mouse , and djungarian hamster ( rodents that can be entrained by daily melatonin administration and in which a high density of melatonin receptors is observed within the scn ) , the mink ( mustela vison ) does not appear to have specific melatonin receptors ( at least 2-iodomelatonin binding sites ) within the scn . newborn syrian hamsters express melatonin receptors in the scn , but the receptor number decreases shortly after birth . young hamsters are entrainable by daily acute melatonin administration , while , in the adult , melatonin is unable to entrain or synchronize , except under particular experimental conditions ( eg , long - term infusions which affect the x ) . since scn - lesioncd hamsters whose rhythmicity had been restored with fetal hypothalamic graft are entrained by daily melatonin injection , it is evident that the chronobiotic effect of exogenous melatonin is the consequence of an action on the clock . this conclusion is supported by the observation that in vivo a melatonin receptor antagonist ( s 22153 ) blocks the phase - advancing effect of melatonin . which receptor subtypes arc involved ? similarly , the most robust entraining response to melatonin , synchronization of developing circadian pacemakers in syrian hamsters by melatonin injections , occurs in the absence of a functional mt2 receptor within the scn this strongly suggests the implication of mt1 receptors . in in vitro experiments in animal models that possess both subtypes , the mechanisms involved appear to be more complex . an acute inhibitory effect on neuronal firing and a phase - shifting effect in the rhythm in electrical activity have been described . in mice with a targeted deletion of the mt1 receptor , the acute inhibitory effect of melatonin was abolished , while the phase - shifting effect remained intact . however , this phase shift disappears when the mt2 antagonist 4-phenylpropionamidotctraline ( 4p - pdot ) is added . this suggests that either a low density of mt2 receptors is still capable of producing a phase shift or that an as yet unidentified melatonin receptor subtype is involved . in contrast to previous studies , van den top et al have recently demonstrated the absence of a specific window of sensitivity for melatonin to inhibit scn neuronal activity . this lack of a window of sensitivity contrasts with the phase - shifting effect of melatonin , and indicates that the cellular mechanisms involved in the acute inhibitory effect and in the phase - shifting effect of melatonin are distinct . this may be related to the two types of effects observed in vivo after daily 8 or 16 h melatonin perfusions described above . the presence of mt1 and/or mt2 melatonin receptors appears to be a necessary condition for the chronobiotic effect of melatonin . however , if these high - affinity melatonin receptors were the only mechanism involved , it would be difficult to explain why a pharmacological dose of melatonin is needed . although a strong modulator role of exogenous melatonin on serotonin ( 5-hydroxytryptamine ) 5-ht receptor - mediated responses has been reported , the 5-ht system does not appear to be crucial to the effects of melatonin on circadian rhythms . exogenous melatonin is known to be able to influence , directly or indirectly , the phase and/or the period of the circadian clock . in terms of treatments , this means that exogenous melatonin ( or any agonist ) can be used as a pharmacological tool to manipulate sleep - wake cycle and other circadian rhythms ( chronobiotic properties ) . it has long been known that administration of melatonin can entrain free - running activity rhythms in rodents . entrainment means that the period of the observed rhythm must adjust to , and equal , the synchronizer ( zeitgeber ) cycle ( t ) , and a stable phase relation must be established between the rhythm and the zeitgeber cycle . this synchronization process occurs through daily phase shifts . administering melatonin for a series of t values , 24 h , 23 h 50 min , 23 h 45 min , 23 h 35 min , and 23 h 25 min has led to the definition of the limiting phase advance value to which the rat activity rhythm entrains to melatonin at 35 min . the entrainment limits found in this study correspond quite well to the maximum daily phase shift values defined by the melatonin phase response curve , and the magnitude of phase shift responses to a single melatonin injection ( range 15 - 52 min ) . daily acute melatonin administration in the rat thus causes true entrainment as defined by rnright et al . interestingly , when melatonin is administered by daily infusion , the phase angle difference between the entrained rhythm and the zeitgeber ( melatonin ) depends upon the duration of the infusion period . a negative phase angle is observed and its value increases with the duration of the infusion period . moreover , with long infusion times ( 8 h and , more especially , with 16 h ) , melatonin induces a change in the free - running period in the first days , suggesting that melatonin delays the pacemaker each day until entrainment occurs . in other words , with a long duration of infusion , entrainment occurs earlier than predicted by the model based on acute melatonin administration . these observations suggest that , beside its chronobiotic properties , melatonin affects the circadian clock properties ( effect on the period t ? ) . melatonin induced entrainment after a time during which circadian periods were either lengthened ( in a fraction of the animals ) or shortened ( in the others ) . this finding suggests that , to achieve entrainment , melatonin has to induce either a phase delay ( when the period is shortened ) or a phase advance ( when the period is lengthened ) . for example , when rats received a 5 h phase advance of the dark onset in ld conditions , those injected melatonin daily at the new dark onset reentrained with decreased latency ; some of the animals did so by phase delays , whereas others did so by phase advances . melatonin has been reported to entrain hamsters and arvicanthis ansorgei , a diurnal rodent , by inducing phase advances when the free - running period is longer than 24 h and phase delays when the period is shorter than 24 h. all these observations strongly suggest that the effects of exogenous melatonin are complex and depend on the period before entrainment . another potential effect of exogenous melatonin should be considered . a single application of melatonin within the scn , in vivo , induced a long - lasting increase in the amplitude of the nocturnal melatonin secretion . this effect demonstrates that exogenous melatonin is able to sustain the oscillation of the clock and suggests a possible role for endogenous melatonin in mammals . in the experiments reported above , responsiveness to melatonin is restricted to a narrow window of sensitivity , which is generally late in the subjective afternoon , but depends upon the duration of the melatonin signal as well as the previous free - running period . the finding that pinealectomized rats entrain to daily melatonin administration indicates that endogenous melatonin is not necessary for the entrainment effect of exogenous melatonin , for example , by entraining a window of sensitivity to melatonin . consequently , the effects of melatonin administration in vivo appear not to be related to the role of endogenous melatonin on circadian function . this conclusion is reinforced by the observation that to obtain entrainment of the circadian activity rhythm of rodents kept under constant darkness ( dd ) , high doses of melatonin have to be used , independently of the mode of administration . ' ill esc doses of melatonin produce peak serum levels 100- to 1000-fold higher than the endogenous melatonin nighttime levels . the necessity of such a high dose of melatonin is unlikely to be a consequence of its rapid metabolism . appropriate photoperiodic response is indeed obtained when melatonin is administered via a similar subcutaneous infusion system with a dose that mimics the endogenous secretion profile . most likely , this high dose of melatonin is needed because it is an integral part of the response observed . in vitro administration of melatonin can phase shift the firing rate of scn neuron brain slices ( rat and mouse ) . it is principally for this reason that it is generally believed that melatonin mediates these effects through the highaffinity melatonin receptors located within the scn . the high correlation between the density of melatonin receptors within the scn and the ability of daily melatonin administration to entrain the free - running activity rhythm in mammals supports this view . in contrast to the rat , mouse , and djungarian hamster ( rodents that can be entrained by daily melatonin administration and in which a high density of melatonin receptors is observed within the scn ) , the mink ( mustela vison ) does not appear to have specific melatonin receptors ( at least 2-iodomelatonin binding sites ) within the scn . newborn syrian hamsters express melatonin receptors in the scn , but the receptor number decreases shortly after birth . young hamsters are entrainable by daily acute melatonin administration , while , in the adult , melatonin is unable to entrain or synchronize , except under particular experimental conditions ( eg , long - term infusions which affect the x ) . since scn - lesioncd hamsters whose rhythmicity had been restored with fetal hypothalamic graft are entrained by daily melatonin injection , it is evident that the chronobiotic effect of exogenous melatonin is the consequence of an action on the clock . this conclusion is supported by the observation that in vivo a melatonin receptor antagonist ( s 22153 ) blocks the phase - advancing effect of melatonin . which receptor subtypes arc involved ? similarly , the most robust entraining response to melatonin , synchronization of developing circadian pacemakers in syrian hamsters by melatonin injections , occurs in the absence of a functional mt2 receptor within the scn this strongly suggests the implication of mt1 receptors . in in vitro experiments in animal models that possess both subtypes , the mechanisms involved appear to be more complex . an acute inhibitory effect on neuronal firing and a phase - shifting effect in the rhythm in electrical activity have been described . in mice with a targeted deletion of the mt1 receptor , the acute inhibitory effect of melatonin was abolished , while the phase - shifting effect remained intact . however , this phase shift disappears when the mt2 antagonist 4-phenylpropionamidotctraline ( 4p - pdot ) is added . this suggests that either a low density of mt2 receptors is still capable of producing a phase shift or that an as yet unidentified melatonin receptor subtype is involved . in contrast to previous studies , van den top et al have recently demonstrated the absence of a specific window of sensitivity for melatonin to inhibit scn neuronal activity . this lack of a window of sensitivity contrasts with the phase - shifting effect of melatonin , and indicates that the cellular mechanisms involved in the acute inhibitory effect and in the phase - shifting effect of melatonin are distinct . this may be related to the two types of effects observed in vivo after daily 8 or 16 h melatonin perfusions described above . the presence of mt1 and/or mt2 melatonin receptors appears to be a necessary condition for the chronobiotic effect of melatonin . however , if these high - affinity melatonin receptors were the only mechanism involved , it would be difficult to explain why a pharmacological dose of melatonin is needed . although a strong modulator role of exogenous melatonin on serotonin ( 5-hydroxytryptamine ) 5-ht receptor - mediated responses has been reported , the 5-ht system does not appear to be crucial to the effects of melatonin on circadian rhythms . melatonin is produced nocturnally by the pineal gland , in a pattern that reflects the phase and duration of the night . in fact , nocturnal melatonin secretion is a hormonal output signal of the circadian clock able to convey photoperiodic as well as circadian signals to multiple structures and organs possessing melatonin receptors , within the brain or at the periphery . the use of melatonin to control seasonally expressed traits of economic importance ( milk and wood production , etc ) in farm animals is now well documented , and the sites and mechanisms of action involved are beginning to be identified . the exact role of the hormone in the circadian timing system remains to be determined . however , due to the presence of melatonin receptors within the scn itself , exogenous melatonin has been shown to affect the circadian clock in animal models ( chronobiotic effect ) . the observations that , in humans , melatonin improves some circadian - based disorders refer to such properties and will lead to strategies to treat , prevent , or delay such disturbances . the hormone 's physiological functions - and thereby its therapeutic potential - will depend on our knowledge of its mechanism of action . today , the pathways through which temporal information encoded in the melatonin signal is decoded in target tissues , and the phenotypic nature of those target tissues , are not completely understood . experimental work in animal models is still needed to define exactly the therapeutic value of the hormone ( for more perspectives with the use of pharmacological tools based on melatonin receptors and a - antagonists see reviews in references 63 and 92 ) .

melatonin is a hormone synthesized and secreted during the night by the pineal gland . its production is mainly driven by the orcadian clock , which , in mammals , is situated in the suprachiasmatic nucleus of the hypothalamus . the melatonin production and release displays characteristic daily ( nocturnal ) and seasonal patterns ( changes in duration proportional to the length of the night ) of secretion . these rhythms in circulating melatonin are strong synchronizers for the expression of numerous physiological processes . in mammals , the role of melatonin in the control of seasonality is well documented , and the sites and mechanisms of action involved are beginning to be identified . the exact role of the hormone in the diurnal ( orcadian ) timing system remains to be determined . however , exogenous melatonin has been shown to affect the circadian clock . the molecular and cellular mechanisms involved in this well - characterized chronobiotic effect have also begun to be characterized . the circadian clock itself appears to be an important site for the entrapment effect of melatonin and the presence of melatonin receptors appears to be a prerequisite . a better understanding of such chronobiotic effects of melatonin will allow clarification of the role of endogenous melatonin in circadian organization .

Melatonin and seasonal function Melatonin and circadian function Exogenous melatonin and circadian rhythms Sites of action for the effects of exogenous melatonin on the circadian activities Conclusions and perspectives

PMC4908255

india is of a fortune position of having the world 's best breeds of buffaloes for milk production . special attention has to be focused on murrah breed of buffalo whose breed average milk production is about 2200 kg per lactation . however , due to increased prevalence of infections , the realization of their true genetic merit has been hampered . among infectious diseases , mastitis , an inflammatory disease of the mammary gland generally caused by intramammary infections , is the most common , costly , and devastating disease in dairy animals . therefore , attention needs to be focused to study the genes involved in disease resistance , especially for mastitis . genes associated with immune responses of the mammary gland are potential markers because of their importance in mastitis . the toll - like receptor-4 ( tlr-4 ) is an important pattern recognition receptor that recognizes endotoxins associated with gram negative bacterial infections [ 1 , 2 ] . its role in pathogen recognition and subsequent initiation of the inflammatory and immune responses , and highly polymorphic nature in the bovine species , make it a suitable candidate gene for use in marker - assisted selection for enhancing disease resistance in dairy animals . the tlr-4 gene coding region is 2526 bp long consisting of 3 exons and is located on chromosome bta 8 . bovine tlr4 has three exons , exon 1 includes coding base pairs 195 , exon 2 consists of base pairs 96260 , and exon 3 comprises base pairs 2612526 . the whole genomic length is estimated to be approximately 11 kb , of which the first intron comprises about 5 kb and the second is 3 kb . polymorphic studies and nucleotide sequencing of tlr-4 gene have been reported in cattle [ 4 , 5 ] . with the exception of the thesis by sonawane , no such information is available in murrah buffalo . considering the importance of murrah buffalo in milk production , the present study was undertaken to partially sequence the buffalo tlr-4 gene and to detect snp . the animals included in the present study were from the herd of murrah buffaloes maintained at cattle yard of national dairy research institute , karnal , haryana , india . ten ml of blood was collected aseptically by jugular vein puncture in a sterile vacutainer tube containing 15% of 0.12 ml edta solution ( becton - dickinson vacutainer ) . the samples were transported to the laboratory in an icebox and stored at 4c till further processing for dna isolation . the blood samples were centrifuged and dna was isolated from the buffy coat alone using phenol - chloroform method , as described by sambrook et al . with few modifications . quality of dna was checked by electrophoresis by loading 2 l dna on 0.8% agarose in horizontal minielectrophoresis unit using 1xtbe as running buffer at 3040 volts for about one and a half hours . after electrophoresis , the gel was stained with ethidium bromide solution ( 0.5 g / ml ) . dna ( 2 l ) was dissolved in 98 l of double - distilled water and loaded into a 100 l cuvette . optical density ( od ) was determined at wavelengths 260 nm and 280 nm in a uv - vis spectrophotometer against distilled water as blank sample . the sample possessing a ratio of less than 1.7 and more than 2.0 was subjected to proteinase k digestion and dna extracted with phenol chloroform isoamyl alcohol as described previously . the primer pairs for exons 1 and 2 of tlr-4 gene were designed by using the primer 3 plus software , and primers 3 and 4 which are part of exon 3 were used as described by sonawane . primers for tlr 4 gene are as follows : forward 5-catgctgatgatgatggcgcgtg-3 and reverse 5-cgtacgatcactgtacgcaagg-3 for exon 1 , forward 5-ttgttcctaacattagttacc-3 and reverse 5-ctggataaatccagcacttgcag-3 for exon 2 , forward 5-ggctggttttgggagaattt-3 and reverse 5-tgtgagaacagcaacccttg-3 for exon 3.1 , and forward 5-ccagagccgatggtgtatct-3 and reverse 5-cactgaatcaccgggcttt-3 for exon 3.2 . for amplification , 25 l of pcr reaction was prepared by adding each primer , dntps , mgcl2 , 10 pcr assay buffer , dna template , and taq dna polymerase . the amplification was carried out using a preprogrammed thermal cycler ( eppendrof mastercycler ) with the following conditions : initial denaturation of 2 min at 95c followed by 35 cycles of denaturation at 94c , annealing at 55c for primers 1 and 2 , 54c for primers 3 and 4 for 30 sec , extension at 72c each of 1 min 30 sec and lastly the final extension of 7 min at 72c . after pcr amplification , 5 l the pcr product was checked on a 1.5% agarose gel to verify the amplification of target region . the amplified pcr fragments , namely , exon 1 , exon 2 , exon 3.2 , and exon 3.2 of tlr 4 gene were digested with dra i ( 5ttt / aaa3 ) , hae iii ( 5gg / cc3 ) , hind iii ( 5a / agctt3 ) , and hinf i ( 5g / antc3 ) restriction enzymes , respectively . the reaction mixture ( 20 l ) for each enzyme was kept for incubated at 37c for 4 hours . restriction fragments were resolved on 2 - 3% agarose gel horizontal electrophoresis and visualized by ethidium bromide staining . the ethidium bromide was added to the agarose gel of 1 l/100 ml of gel . the agarose gel electrophoresis was performed in 1x tbe buffer at 100 volts for 30 , 60 , and 90 minutes till complete separation and visualization of all fragments of re - digested gene fragments , dna ladder and pcr marker . the restriction - digested gene fragments were visualized on uv transilluminator and photographed with gel documentation system . amplified pcr products were subjected to custom dna sequencing from both ends ( 5 and 3 ends ) . representative samples from each of the variants obtained by rflp analysis were also custom sequenced ( chromous biotech pvt . ltd . , sequence data were edited using the editseq program , and multiple sequence alignments were performed with megalign program of lasergene software , respectively ( dnastar , inc , madison wi , usa ) . the forward and reverse sequences for each pcr fragment were assembled to form contigs of the respective region . the tlr-4 gene sequence of murrah was compared with that of bubalus bubalis ( eu386358 ) sequence to annotate different exonic regions putatively to identify snps in respective region . the partial coding dna sequence of bubaline tlr-4 gene ( exons 1 , 2 , and 3 ) was conceptually translated and compared with that of the bubalus bubalis to detect amino acid changes in buffalo tlr-4 regions included in present study . the contiguous tlr-4 nucleotide sequence was subjected to basic local alignment search ( blast ) at ncbi database to determine the sequence homology with the corresponding regions of other species . the amplified pcr product was checked on 1.5% agarose to verify the amplification of target region . the amplified sizes were estimated as 165 bp for exon 1 , 300 bp for exon 2 , 478 bp for exon 3.1 , and 409 bp for exon 3.2 . polymerase chain reaction - restriction length polymorphism ( pcr - rflp ) analysis of each pcr product was carried out using dra i , hae iii , hind iii , and hinf i restriction enzymes for all 102 animals included in this study system . restriction digestion of amplicon of exon 1 revealed two fragments of 110 and 55 bp exhibiting monomorphic ( bb ) pattern in all the animals under study . however , exon 2 of tlr4 gene did not have any cutting site with dra i. restriction digestion of exon 3.1 resulted in resolution of 5 fragments , identified as aa ( 478 , 350 , 272 , 169 bp ) , ab ( 478 , 350 , 272 , 169 , 74 bp ) , and bb ( 272 , 169 , 74 bp ) genotypes . tlr4-exon 3.2 exhibited aa ( 409 bp ) ab ( 409 , 246,163 bp ) and bb ( 246,163 bp ) genotypes with this restriction enzyme . pcr - rflp of exon 1 with hae iii re yielded two genotypes ab ( 165 , 122 , and 43 bp ) and bb ( 122 and 43 bp ) . exons 2 and 3.1 of tlr4 gene did not have any cutting site with hae iii re . exon 3.2 exhibited aa ( 409 and 309 bp ) , ab ( 309 , 200 , 142 , and 100 bp ) , and bb ( 200 , 142 , and 100 bp ) genotypes . pcr - rflp analysis of tlr4 gene using hind iii restriction enzyme did not reveal any cutting site . pcr - rflp analysis of exon 1 of tlr4 gene using hinf i restriction enzyme yielded two fragments of 110 bp and 55 bp size . exon 2 of tlr4 gene did not have any cutting site with hinf i re . the only genotype exhibited by exon 3.1 of tlr-4 was bb with 291 and 187 bp restriction fragment size . for exon 3.2 , genotypes with restriction fragment were identified as aa ( 409 , 308 , and 292 bp ) , ab ( 308 , 292 , 200 , 136 , and 100 bp ) , and bb ( 200 , 136 , and 100 bp ) . the present findings of murrah buffalo could not be compared with other studies , as no such report on buffalo is available in the literature . in a recent study by sonawane in the same buffalo herd , three genotypes aa , ab , and bb with variable frequencies using alu i , bsp 1286 i , and bshkai restriction enzymes were reported . however , exon 2 in that study was also observed as highly conserved part of the gene . hence , no cutting site was observed using 7 enzymes ( 3 res by sonawane , and 4 in the present study ) . sharma et al . reported cc , cg and gg , genotypes in the promoter region ( p 226 ) of holstein cattle . wang et al . reported moderate occurrence of polymorphism with alui in chinese simmental , holstein , and sanhe cattle . nucleotide sequencing of amplified fragments of tlr-4 gene of buffalo was performed ( figures 1 , 2 , 3 , 4 , and 5 ) . the coding dna sequence of bubaline tlr4 gene compared with that of this sequence was compared to the reported sequence of bubalus bubalis with ncbi accession number eu386358 . the sequence obtained for murrah was compared and aligned custom sequenced using the megalian program of dnastar software . amplified regions of the 4 contig regions clustal w multiple alignments with bubalus bubalis sequence revealed a total of 12 bp changes , one in exon 1 and 11 in exon 3 . multiple alignment revealed a total of 12 mutations : 1 in exon1 and 11 in exon 3 . out of these 12 mutations , six were nonsynonymous resulting in change in threonine to methionine , valine to arginine , tyrosine to serine , glutamine to histidine , and aspartic acid to glycine ( at two positions ) ( figure 6 ) . sequence analysis revealed 12 snps in the coding ( exonic ) region of tlr-4 gene given in table 1 . the coding dna sequences of murrah tlr-4 gene ( exon 1 , 2 , and 3 ) were conceptually translated and compared with those of bubalus bubalis reported sequences ( ncbi accession number eu386358 ) . at position 75nt of exon 1 , only one snp ( t to c ) however , a total of 11 snps have been identified in exon 3 at nucleotide positions 311 , 315 , 316 , 318 , 386 , 401 , 411 , 551 , 555 , 636 , and 994 . only five of these nucleotide changes result into changes in amino acids leading to nonsynonymous snps . in the only report available till date , sonawane has reported a total of six snps , out of which 4 are nonsynonymous , two each in exons 1 and 3 . however , in holstein cattle , sharma et al . have reported 3 snps : 1 in promoter region ( p-226 ) and 2 in exon 3 ( 1656 and 2021 ) . wang et al . , 2007 have reported 31 snps scattered through the 5 flanking region to exon 3 . the contiguous tlr-4 nucleotide sequence was subjected to basic local alignment search tool ( blast ) at ncbi database to know the sequence homology with the corresponding regions of other species . it revealed 97% , 97% , 99% , 98% , and 80% homology with bos indicus , bos taurus , ovis aries , capra hircus , and homo sapiens respectively . sequence alignment and homology across species using basic local alignment search tool ( blast ) analysis revealed 97% , 97% , 99% , 98% , and 80% sequence homology with bos taurus , bos indicus , ovis aries , capra hircus , and homo sapiens , respectively . in conclusion , nucleotide sequencing of the amplified fragment of tlr-4 gene of murrah buffalo revealed twelve snps : 1 in exon1 and 11 in exon 3 . murrah is an indigenous buffalo breed , and the presence of the nonsynonymous snp is indicative of its unique genomic architecture . sequence alignment and homology across species using basic local alignment search tool ( blast ) analysis revealed 97% , 97% , 99% , 98% , and 80% sequence homology with bos taurus , bos indicus , ovis aries , capra hircus , and homo sapiens , respectively .

toll - like receptor-4 ( tlr-4 ) has an important pattern recognition receptor that recognizes endotoxins associated with gram negative bacterial infections . the present investigation was carried out to study nucleotide sequencing and snp detection by pcr - rflp analysis of the tlr-4 gene in murrah buffalo . genomic dna was isolated from 102 lactating murrah buffalo from ndri herd . the amplified pcr fragments of tlr-4 comprised of exon 1 , exon 2 , exon 3.1 , and exon 3.2 were examined to rflp . pcr products were obtained with sizes of 165 , 300 , 478 , and 409 bp . tlr-4 gene of investigated murrah buffaloes was highly polymorphic with aa , ab , and bb genotypes as revealed by pcr - rflp analysis using dra i , hae iii , and hinf i res . nucleotide sequencing of the amplified fragment of tlr-4 gene of murrah buffalo was done . twelve snps were identified . six snps were nonsynonymous resulting in change in amino acids . murrah is an indigenous buffalo breed and the presence of the nonsynonymous snp is indicative of its unique genomic architecture . sequence alignment and homology across species using blast analysis revealed 97% , 97% , 99% , 98% , and 80% sequence homology with bos taurus , bos indicus , ovis aries , capra hircus , and homo sapiens , respectively .

1. Introduction 2. Materials and Methods 3. Custom DNA Sequencing 4. Results and Discussions 5. Analysis of Sequencing Data 6. SNP Identification 7. Sequence Alignment and Homology Across Species 8. Conclusion

PMC4004252

monomeric human topoisomerase 1b ( top1 ) regulates dna topology throughout key cellular events such as dna replication and gene transcription . eukaryotic top1 relaxes both positively and negatively supercoiled dna and is an established anticancer drug target ( its inhibition initiates apoptosis and hence tumor regression ) . recent single - molecule nanomanipulation and molecular dynamics studies of top1 in the presence and absence of inhibitors and the originally proposed catalytic cycle of top1 may be used to construct a current view of the enzyme s four - step cycle ( figure 1 ) . drugs that block top1 fall into two distinct classes : ( 1 ) well - characterized interfacial poisons ( ifps ) and ( 2 ) less common catalytic inhibitor compounds ( cics ) . currently , all dna - intercalating ifps arrest dna strand religation by non - covalent binding at the nick site of the top1dna cleavage complex , poisoning the enzyme midcycle . known ifps include camptothecin ( cpt ) and its analogues and synthetic compounds , e.g. , indolocarbazoles , indenoisoquinolines , dibenzonaphthyridones , and aromathecins . some minor - groove binders that engage top1 s dna substrate below the nick site , e.g. , hoechst 33258 and 33342 , prevent strand religation and are non - interfacial top1 poisons . the rational design of new ifps and conceptual understanding of how established ifps work is underpinned by x - ray data for the dna cics may operate by blocking two key steps in the enzyme s catalytic cycle : substrate binding or covalent cleavage complex formation . compounds inhibiting the first step ( cic1 ) are either conventional competitive inhibitors ( binding to top1 ) or unconventional competitive inhibitors ( binding to dna ) . examples of the former are unknown , but unconventional competitive inhibitors exist and are either dna intercalators or minor groove binders or both . step 2 catalytic inhibitors ( cic2 ) are currently rather obscure ; one recently described indolizinoquinoline-5,12-dione derivative , cy13i , possibly fits this descriptor . of relevance to this study , dna - binding au porphyrins were classified as top1 catalytic inhibitors , while other au complexes were initially misassigned as top1 ifps and hence reclassified as catalytic inhibitors . the correct assignment of a compound s mechanism of action ( moa ) with top1 is not straightforward . the inherent problem is that inhibition of supercoiled dna relaxation by top1 alone does not distinguish between the actions of cics and ifps nor does it distinguish between conventional and unconventional competitive inhibition . since cic s do not trap top1-nicked dna , dna damage by this class of compounds is likely to be lower than that caused by ifps . the paucity of top1 cics coupled with their anticipated reduced genotoxicity relative to ifps creates significant opportunities for drug discovery . here we report on a new class of cytotoxic macrocyclic au top1 cics ( figure 1 ) and precise delineation of the moa of the lead compound , 3 . ( a ) illustration of key events in the catalytic cycle of human top1 . step 1 : top1 binds supercoiled dna ( scdna ; 5-ta-3 dinucleotide pair as target ) . step 2 : nucleophilic attack of the 3-phosphate linking the ta pair ( scissile strand ) by y723 ( catalytic tyrosine residue ) affords a covalent dna step 3 : the intrinsic torque stored in scdna drives ratchet - like rotation about the non - scissile strand until strand religation occurs with concomitant release of y723 . the relaxed dna ( rdna ) is then released by the enzyme in step 4 . interfacial poisons ( ifps ) such as camptothecin ( cpt ) bind the nick site via 5-ta-3 intercalation and h - bonding to top1 to form a ternary drugscdna cics operate differently by either blocking substrate recognition by top1 ( type 1 competitive inhibitor , cic1 ) or , in principle , preventing the formation of the covalent cleavage complex by blocking the nucleophilic attack of the scissile strand by y723 ( type 2 competitive inhibitor , cic2 ) . ( b ) structures of new cytotoxic pyrrole - based au macrocycles 15 , free base macrocycle 6 , and the ni analogue of 1 , compound 7 . compounds 15 reflect a design evolution over our recently patented class of cytotoxic bis(pyrrolide - imine ) au chelates . specifically , we have employed macrocycles to enhance the redox and chemical stability of the metal ion in conjunction with a quinoxaline ring to augment dna intercalation . direct metalation of the macrocycle ( route a , figure s2 ) was only successful for 1 and 5 ( i.e. , those macrocycles bridged by a propyl chain ) . for compounds 24 with slightly more elaborate alkyl groups linking the imine nitrogen atoms , a metal - templated cyclization had to be employed ( route b ) . more specifically , the diamine bridge required to effect cyclization of the macrocycle was added after au - binding by the bis(pyrrole - aldehyde ) precursor . ( although uncommon , au ions reportedly template aldehyde and amine condensations . ) we were able to elucidate the structures of compounds 1 , 2b , and 3 by single crystal x - ray diffraction ( table s1 ) despite the ordinarily challenging morphology of the crystals ( fine , brick - red needles ) . because of the high degree of similitude in the structures of 13 , particularly the au ion coordination geometry and macrocycle conformation and the appreciable cytotoxicity of 3 ( vide infra ) , the following discussion is illustrated with selected , typical ion pairs from the asymmetric unit of 3 ( figure 2 ) . the au ions in salts 1 , 2b , and 3 are nominally square planar with au the au npyrrole distances average 1.98(3 ) for all nine cations and are 1.5% shorter than the au nimine bonds , which average 2.01(6 ) ( table s2 ) . this reflects the fact that the pyrrole groups are anionic -donors with more acute c n c angles than the corresponding c = n c angles of the imine donors . the coordination group distances are comparable to those of other au complexes ( au n : 1.9282.216 ) . the average npyrrole au npyrrole and nimine au nimine bond angles for the propyl - bridged systems 1 and 2b are 99.6(8) and 96.5(5) , respectively . for the six independent cations of 3 , these angles average 97.1(9) and 99.2(6). the larger seven - membered chelate ring of 3 accounts for the somewhat more obtuse mean nimine au nimine bond angle . the cation conformations of 13 deviate mildly from planarity mainly in response to crystal packing constraints . in each structure , the cations form -stacked dimers with head - to - tail ( figure 2 ) or oblique geometries ( figures s8s14 ) characterized by interplanar spacings of 3.4 , as found in other -stacked polyaromatic compounds . there are no aurophilic auau contacts between cations ( auau distances > 5 ) . the au macrocycle pairs within dimers of 13 exhibit overlaps ranging from 74% in 3 to 89% in 1 . importantly , the -stacking proclivity of the cations highlights their potential as dna intercalators . view of the x - ray structure ( 100 k ) of two of six independent ion pairs from salt 3 ( 50% thermal ellipsoids for non - h atoms ; h atoms are shown as capped cylinders ) . atom color code : gray , c ; lilac , n ; orange , p ; green , f ; gold , au ; pale blue , h. crystallographic details for 1 , 2b , and 3 are given in the supporting information . selected mean bond distances ( ) and angles ( ) for all six independent cations of 3 : au npyrrole , 1.98(1 ) ; au nimine , 2.02(2 ) ; c = nimine , 1.30(3 ) ; npyrrole au the mean plane separation ( 3.4 ) reflects notable -stacking for both the head - to - tail and oblique dimers of 3 . the affinity constants , ka , for non - covalent binding of 15 to calf - thymus dna ( ctdna ) were determined by fluorometric titrations involving the displacement of intercalated ethidium bromide ( eb , figure 3a ) . from the loss of eb fluorescence ( 614 nm ) with increasing concentration of 15 , ka values ranging from 2 10 to 4 10 m bp were obtained and are similar in magnitude to that reported for a gold(iii ) porphyrin ( 3 10 m bp ) . the ka values for 15 showed no independent linear correlation with either the steric bulk of the macrocycle s alkyl bridge or the lipophilicity of the cation . however , analysis of ka as a function of both variables concurrently ( figure 3b ) gives a significant three - dimensional bivariate linear correlation and confirms that cations with a small alkyl bridge and high lipophilicity , e.g. 5 , exhibit the highest ka values . the correlation itself suggests that 15 are dna intercalators since not only are the ka values determined by competitive displacement of a known dna intercalator ( eb ) but also the monotonic increase in ka with increasing lipophilicity of the compounds clearly reflects binding principally within the relatively non - polar intrahelical space of the duplex dna target ( i.e. , between -stacked bases ) . to confirm the intercalation data and examine possible roles for the metal ion , electrophoretic mobility shift assays ( emsas ) , thermal denaturation , and dna unwinding experiments were carried out . regarding the first of these tests , figure 3c shows that 1 induces a greater mobility shift for a plasmid substrate ( phot1 , lanes 59 ) than the archetypal reference compound for dna intercalation ( eb , lanes 24 ) , despite the comparable ctdna affinity constants for the two compounds ( eb , ka = 5(1 ) 10 m bp ; compound 1 , ka = 4.0(4 ) 10 m bp ) . the large mobility shift induced by 1 note that supercoiled plasmid dna is affected most upon binding of 1 ( figure 3d ) , as evidenced by the 11% mobility shift relative to nicked - open circular dna ( noc dna , 5% shift ) and very marked band broadening apparent at a concentration of only 5 m ( lane 7 ) . in the case of a negatively supercoiled plasmid with intact double strands , the linking number describing the dna topology , lk , must remain constant irrespective of the extent of local unwinding ( i.e. , change in twist , tw ) induced by the intercalator . to keep lk constant , wr , which reflects supercoiling of the plasmid , must adjust according to eq 1:1for au macrocycle 1 , intercalation of negatively supercoiled phot1 is expected to reduce the number of supercoils and thus the macromolecule s supercoil density , thereby significantly impeding its mobility in the gel matrix ( as observed ) . the three - dimensional plot displayed in figure 3d further highlights the dna distribution with increasing in lanes 58 and specifically emphasizes the appearance of a third , non - migratory form of dna ( form iii , lanes 7 and 8) . two possible explanations for the appearance of this immobile dna species at higher concentrations of 1 are : ( i ) aggregation of the dna1 adduct(s ) occurs , trapping the dna in the well , or ( ii ) full charge - neutralization of the dna takes place . however , the latter explanation seems unlikely since each adjacent dinucleotide pair in dsdna is bridged by two phosphate groups , each with a formal charge of 1 . intercalation of a monocationic intercalator can thus only neutralize 50% of the total charge on the dna duplex even if every adjacent dinucleotide pair non - covalently binds to one au macrocycle , which is improbable . finally , the apparent disappearance of the dna in lane 9 ( = 50 m ) signals either reverse migration of all dna1 adducts or complete saturation of the eb binding sites by 1 so that staining of the gel for dna visualization is ineffective . the latter explanation is the more likely given that formation of a cationic dna1 adduct ( as would be required for reverse migration of the dna ) is physically highly improbable . ( a ) displacement of intercalated eb from ctdna by 3 studied by emission spectroscopy ( 298 k , ph 7.0 , 15% dmso - tris / hcl buffer , 15 m ctdna , 15 m eb ) . inset : determination of at 50% loss of eb fluorescence ( c50 ) ; c50 is used to determine the ctdna affinity constant , ka , of 3 . ( b ) graph of ka for 1 , 2a , and 35 as a function of the steric bulk of the macrocycle s alkyl bridge and the hydrophobicity of the au complex , log(po / w ) ( table s3 ) . the surface is the best - fit bivariate linear regression function to the data . ( c ) emsa of selected compounds with supercoiled ( sc , form i ) phot1 plasmid dna ( dna , 31.3 ng / well ; tbe buffer , ph 7.8 ) ; some nicked - open circular ( noc , form ii ) dna is present . the lanes contain phot1 plasmid dna ( lane 1 ) , increasing concentrations of eb ( lanes 24 ) , 1 ( lanes 59 ) , 6 ( lanes 1012 ) , and 7 ( lanes 1315 ) . ( d ) two- and three - dimensional deconvolution of the dna bands in lanes 58 of the emsa gel shown in part ( c ) . collectively , the emsa data for 1 reflect intercalative binding of dsdna by the au macrocycle . this conclusion was further tested by concurrent analysis of the metal - free macrocycle ( compound 6 ) and the ni analogue of 1 ( compound 7 ) ; neither induces a dna mobility shift nor in fact displaces eb from dna in solution . the au ion is thus obligatory for dna intercalation by 15 . that association depends on the presence of au reflects electrostatic binding of the intercalator to the dna ( as neither 6 nor 7 are charged ) and a pivotal iondipole interaction formed between the au ion and a carbonyl oxygen atom of thymine ( vide infra ) . because the mode of interaction of the au macrocycles with dna has to be established unambiguously to determine the mechanism of action underpinning the cytotoxicity of 2a and 3 ( vide infra ) , we performed two additional tests that unequivocally support an intercalative dna binding mode for the compounds ( figure 4 ) . in the first experiment , thermal denaturation ( melting ) of a linear 291-bp dna duplex ( an amplicon from exon 15 of the human actn3 gene ) was studied as a function of the identity and concentration of the added compound ( table s12 ) . from figure 4a , b , the melting point , tm , increased in sigmoidal fashion with increasing concentration of 13 , consistent with the enhanced thermal stability that is expected to accompany uptake of an intercalator . significantly , no change in tm was observed for 6 over the full concentration range . the values of tm ( the change in melting point relative to the control ) were + 4.65 , + 3.90 , and + 4.40 c for compounds 13 , respectively , when measured at their highest concentrations ( 50 m ) . from figure 4b , the dose response function for 1 gives the saturating value of the melting point increase , tm = + 5.1(4 ) c . the tm values determined here are in good agreement with those reported for the metallointercalators [ co(pic)2(dppz ) ] ( + 4.4 c ) , where pic = picolinate and dppz = dipyrido[3,2-a:2,3-c]phenazine , and [ ru(nh3)4(dppz ) ] ( + 5.2 c ) as well as organic protoberberine derivatives ( + 2.9 to + 6.4 c ) . other dppz - based metallointercalators , however , exhibit tm values as large as + 14 c . ( a ) hrm curves for a linear 291-bp dsdna fragment ( ph 8.4 ) derived from the human actn3 gene as a function of the concentration of 1 . the plot reflects the negative first derivative of the fluorescence intensity ( df / dt , 510 nm ) from the dna - intercalating reporter dye ( cybr green ) used to monitor strand separation . ( b ) plot of the melting temperatures , tm , against the concentrations of three au macrocycles and the metal - free macrocycle 6 . the zero - slope fit for 6 has an intercept , tm , of 83.0(1 ) c and reflects the mean value of tm for the amplicon ( since 6 does not interact with the dna ) . the dose response function for 1 is : tm = tm + ( tm tm)/(1 + ( c / ec50 ) ) , where c is the molar concentration of 1 . the fit parameters were : tm = 82.95(9 ) c ; tm = 88.1(4 ) c ; ec50 = 9(2 ) 10 m ; p = 0.9(1 ) ; = 0.013 ; r = 0.996 . lane 1 , supercoiled phot1 plasmid dna ; lane 2 , relaxed phot1 plasmid ( effected by incubation with 10 units of top1 for 30 min at 37 c ) ; lanes 35 , increasing concentrations of the intercalator control m - amsa added to relaxed phot1 ; lanes 614 , increasing concentrations of compounds 1 , 7 , and 6 added to relaxed phot1 as indicated . abbreviations : noc , nicked - open circular dna ; sc , supercoiled dna ; rx , relaxed ; ti , dna topoisomer bands ; m - amsa , 4-(9-acridinylamino)methanesulfon - m - anisidide . several factors will determine the measured value of tm ( beyond the identity of the primary intercalating group ) . these include the nature of the ancillary ligands , the base sequence of the dsdna substrate ( since this governs the density of binding sites for base pair - specific intercalators ) , the solution conditions ( ph , buffers , salts ) , and the experimental method . the method used here evidently affords reliable data over a wide concentration range , permitting measurement of tm . however , as shown in figure 4a , some caution in evaluating the data is required at higher compound concentrations when the signal is weak ( df / dt < 0.1 ) . the loss of signal intensity in the experiment reflects two main processes : ( 1 ) the release of cybr green ( an intercalating dye that binds at base pairs in dsdna via minor groove entry ) upon strand separation and , consequently , quenching of its emission by torsional motion in its non - intercalated state . this is the physical basis underpinning melt analysis with the high - resolution melt ( hrm ) method available in commercial real - time pcr machines . ( 2 ) competition between the dye and intercalator for binding sites along the length of the dsdna substrate . this is evident from the dose - dependent decrease in the emission signal from the dye at a fixed temperature in figure 4a and accounts for the vanishing signal at high concentrations of 13 . further , from the data for 6 , the irremissibility of the au ion for dna intercalation is again evident . in the second experiment , we used a standard dna unwinding assay designed to identify intercalators from their interaction with dna in the presence of top1 ( figure 4c ) . the method was first described by hsiang et al . in 1985 and elaborated on by pommier et al . in 1987 . in our experiment , phot1 plasmid dna was first relaxed by incubation with 10 units of top1 for 30 min at 37 c ( lane 2 ) . increasing concentrations of the test compounds were then added , and the solutions incubated for a further 30 min interval at 37 c prior to workup and analysis . the dna intercalator control m - amsa gave the expected gaussian distribution of dna topoisomers ( characterized by a linking number change , lk , of 1 ) at a concentration of 50 m after workup . the same topoisomer band distribution was observed at a concentration of only 5 m in the reaction with 1 ( lane 7 ) , consistent with 1 being a more potent dna intercalator than m - amsa . at a higher concentration ( 50 m ) , the reactions with the ni analogue of 1 ( compound 7 ) and the metal - free macrocycle 6 confirm that neither intercalate dna since the signature dose - dependent gaussian distribution of topoisomer bands is noticeably absent in lanes 914 . similar results were obtained running the reaction the other way ( i.e. , starting with supercoiled phot1 ; figure s54 ) . the observations for 1 in lane 8 merit further reflection . as discussed in the literature and supporting information , the method used is incapable of determining whether the scdna product obtained at high concentrations of the intercalator is : ( 1 ) positively or negatively supercoiled or ( 2 ) the result solely of intercalator - induced torque or , alternatively , inhibition of top1 . from lane 8 in figure 4c , it is therefore possible that 1 may be an inhibitor of top1 , but this can not be concluded with certainty in the absence of definitive mechanistic tests ( e.g. , tests that can discriminate between an ifp and a catalytic inhibitor of the enzyme ) . compounds 15 were prescreened by the national cancer institute ( nci , bethesda , md ) against their panel of 60 human cancer cell lines ; 2a and 3 were sufficiently cytotoxic to warrant full five - dose screens and 3 proceeded to in vivo hollow - fiber studies ( figures s35s43 , table s6 ) . the mean ic50 value across all cell lines for the au macrocycle is 16(7 ) m ; the large standard deviation reflects considerable variation in the cytotoxicity of the compound across each group of cell lines ( as highlighted by the radar plot of figure 5 ) . leukemia , central nervous system ( cns ) , and colon cancer were the most sensitive cancer types to 3 . importantly , a total of 19 ( out of 57 ) cancer cell lines had ic50 values for compound 3 that were < 5 m , accounting for selection of 3 by the nci for in vivo hollow - fiber assays ( table s7 , figures s42 and s43 ) . the five most sensitive cell lines were nci - h522 ( non - small lung cancer , ic50 = 280 nm ) , rxf-393 ( renal cancer , ic50 = 1.3 m ) , sf-268 ( cns cancer , ic50 = 1.4 m ) , sw-620 ( colon cancer , ic50 = 1.5 m ) , and lox - imvi ( melanoma , ic50 = 1.7 m ) . in the nci s non - tumored animal toxicity assay with female athymic nude mice ( figure s42 ) , compound 3 was remarkably well tolerated as reflected by a 100% survival rate after 17 days for doses ranging from 100 to 400 mg kg dose ( intraperetoneal injection ) . despite these encouraging data , 3 was insufficiently cytotoxic in the majority of the cell lines tested in the nci s hollow fiber cytotoxicity assay to warrant progression to the next investigational phase ( tumor xenograft studies in live mice ) . notwithstanding the lower than desired activity of 3 in the hollow fiber assays , we believe that 3 and its structural congeners represent an important group of compounds that have the potential to be developed into novel metallodrug lead candidates with relatively low side - effects because of the in vivo stability of the macrocyclic au complex . going forward , absorption , distribution , metabolism , and excretion studies are clearly warranted in the case of 3 and may well delineate the factors that reduce the efficacy of the compound in live animals . abbreviations : n , number of cell lines within each cancer category ; gi50 , compound concentration effecting 50% growth inhibition ; ic50 , compound concentration effecting 100% growth inhibition ; lc50 , compound concentration that induces 50% cell death . estimated standard deviations are given in parentheses ; large values indicate variable susceptibility of a specific group of cell lines to the test compound ( see figure s41 ) . ( a ) radar plot of log(gi50 ) values vs cancer cell line from the nci-60 cytotoxicity screen for 3 against their panel of 60 human cancer cell lines . comparative data for cisplatin , camptothecin ( cpt ) , and 5-fluorouricil ( 5fu ) are shown to illustrate the cell - line dependent response to the drugs as well as their effective cytotoxic range [ mean log(gi50 ) values response function for 3 with the cns cancer cell line u251 ( the relevant gi50 value is indicated by the orange arrow on the radar plot ) . ( b ) hierarchical cluster analysis ( group average method , minkowski distances ) of the gi50 data for 3 with analogous data for 26 anticancer drugs with known mechanisms of action ( data taken from the nci database ) . compound 3 clusters with camptothecin ( a top1 ifp ) . since release of an exogenous metal ion by a ligand system in a live animal is often the cause of acute toxicity , an important question to answer prior to determining the moa of a metallodrug candidate is how stable the compound is in aqueous solution , especially in the presence of cellular reducing agents such as glutathione . we found that the au macrocycles of this study were both redox stable and essentially non - aggregating under biologically pertinent conditions ( figures s15 and s16 ) , which might at least partly explain the fact that 3 was particularly well - tolerated in mice and essentially non - toxic even at the highest test concentration of 400 mg kg . from the graph of log(gi50 ) values for 3 against all 60 tumor cell lines ( figure 5a ) , compound 3 is comparable to cisplatin and midway between the limits of highly and mildly cytotoxic compounds such as topotecan ( a top1 ifp ) and 5-fluorouricil ( a clinically deployed antimetabolite ) , respectively . in order to identify the moa and hence the cellular target(s ) of 3 , the gi50 data for 3 were compared with equivalent data for 26 compounds of known moa from the nci s database using hierarchical cluster analysis ( figure 5b ) . the cytotoxicity profile of 3 is clearly akin to that of cpt , the archetypal top1 ifp . although the statistical data strongly suggest that 3 targets top1 in tumor cell cultures , they do not prove that 3 is an ifp of top1 . unambiguous determination of the moa is essential to definitively assign 3 as a top1 ifp or top1 cic . cyril and muller s recent solid - phase enzyme inhibition assay capable of distinguishing between top1 ifps and cics was used to elucidate the moa of 3 ( figure 6a ) . . added ifps trap dna covalently bound to top1 during enzyme turnover , which is subsequently detected by an enhanced fluorescence signal from the dna - sensing dye picogreen . cics of top1 do not result in covalently trapped dna , favoring diminished emission from the reporter dye with increasing compound concentration . at concentrations of 100 m , 14 all give a negative readout ( picogreen relative fluorescence ) akin to the signal obtained from the cic control ( mitoxantrone , mtx ) . from this assay , 14 are clearly catalytic inhibitors of top1 at higher concentrations . this result was confirmed for 3 using a novel assay designed to trap top1dna complexes using ultra - high enzyme : dna ratios ( figure 6b ) . in lanes 15 , increasing [ top1 ] at a fixed [ dna ] in the absence of nacl ( ensuring a high dna affinity for top1 ) increases the yield of noc dna in the reaction . this is the expected result as we previously reported that endogenous top1 may cluster at catalytic sites on genomic dna in situ . importantly , at high [ top1 ] and in the presence of an ifp like cpt , multiple single - strand cleavages afford double - strand breaks and thus linear dna after workup . an ifp of top1 is therefore identified by its ability to generate linear dna in the assay . lanes 1116 confirm that 3 is not an ifp of top1 ( no linear dna ) ; rather , the dose - dependent drop in [ noc dna ] reflects catalytic inhibition of top1 by 3 . from figure 6a , both 1 and mtx give a similar ( positive ) readout to cpt at doses of 0.1 and 1 m , suggesting that they are top1 ifps at low concentrations . the switch to catalytic inhibition of top1 occurs at higher doses ( 110 m ) . this was confirmed for 1 by integrating the dose - dependent linear dna band intensities in a gel equivalent to that shown in figure 6b . from figure s56 , the concentration of linear dna increases with increasing up to 500 nm , thereafter exhibiting a sigmoidal decrease consistent with catalytic inhibition of top1 at higher doses . dual - mode inhibition of topoisomerases is well - known for top2 and is equally feasible , though rarely observed , for top1 . compounds such as doxorubicin target the dna nick - sites in the enzyme dna covalent cleavage complex at low doses , poisoning the enzyme , but intercalate the dna substrate at higher concentrations , engendering catalytic inhibition . mechanistically , a large association constant , ka1 , exists for formation of the ternary drugdna enzyme covalent cleavage complex , while a smaller association constant , ka2 , exists for intercalation of the enzyme s dna substrate . because the two assays used here each permit a distinction to be made between top1 ifps and catalytic inhibitors in a single experiment , they are ideal for detecting dual - mode inhibitors . consequently , 1 can be firmly assigned as a dual - mode ( ifp - cic ) top1 inhibitor . compounds 2a4 , in contrast , are simple cics . in the conventional top1 dna relaxation assays ( topogen , inc . ; figure 6c ) , 3 showed marked inhibition of the enzyme at 500 nm and complete inhibition of the enzyme at a dose of 5 m ( lanes 9 and 10 ) . when compared with the activity of the dna intercalator control m - amsa ( lanes 1315 ) , compound 3 engenders equivalent inhibition of the enzyme at one - tenth of the concentration . collectively , the data in figure 6 demonstrate that the au macrocycles are top1 cics . furthermore , we estimate that the method - dependent ic50 values for 3 are 49(2 ) nm and 9.2(5 ) m from the assays in figure 6c , b , respectively ( figures s44 and s45 ) . notably , these assays neither distinguish between conventional ( drug binds enzyme ) and unconventional ( drug binds substrate ) catalytic inhibition of top1 nor between type 1 and 2 cics of the enzyme . however , since the dna affinity of 15 has been established with certainty , the compounds may be tentatively assigned as unconventional cics of top1 . ( it is important to note that concrete assignment of the above mechanism requires an independent experiment to prove that the compounds do not bind to the enzyme itself ; vide infra . ) enzyme target and mechanism assignment . ( a ) relative fluorescence intensity from dna - bound picogreen ( 525 nm ) vs the concentration of two drug standards ( cpt and mitoxantrone , mtx ) and 14 . the assay measures covalent top1dna trapped by the test compound , distinguishing between ifps and cics . the data are corrected for background emission ( range indicated by the dashed lines ) . ( b ) proof of catalytic inhibition of top1 by compound 3 using a gel - based product - trapping assay ( high enzyme : dna ratio , 0 mm nacl , [ phot1 ] = 31.3 ng / well in all reactions ) . lanes 15 : enhanced yield of nicked - open circular dna ( noc dna ) with increasing [ top1 ] . lanes 610 : effect of increasing [ cpt ] from 0.5 to 50 m ( 100 units of top1 ) . lanes 1116 : catalytic inhibition of top1 ( 100 units ) with increasing attenuates the yield of noc dna ( linear dna , l , is essentially absent above a dose of 1 m ) . ( c ) standard scdna relaxation assay for 3 and the dna - intercalator m - amsa . lanes 13 : phot1 dna alone , dmso control , and top1-mediated relaxation of the substrate ( no inhibitor present ) , respectively . lanes 412 : inhibition of top1 with increasing ( complete inhibition occurs at 5 m ) . m - amsa only completely inhibits top1 at 50 m ( lanes 1315 ) . finally , since hierarchical cluster analysis of the nci-60 data for 3 indicated a moderately close link between the in vitro cellular target(s ) of the au macrocycle and zorubicin ( a top2 poison ) , we evaluated the compound s ability to inhibit top2. the results ( figure s55 ) indicate that 3 is a weak catalytic inhibitor of the enzyme , as evidenced by an inhibitory effect commencing only at a relatively high compound dose of 50 m . since no linear dna was detected in the assay , compound 3 evidently , 3 does not target type ii topoisomerases to the same extent as top1 . from this if top1 s dna substrate is targeted by 3 , then where does intercalative binding of 3 occur ? one can not naively assume that because 3 is a type 1 cic of top1 ( figure 1 ) , it binds to precisely the same nucleotide sequence as the enzyme . we have used a conformational search strategy with an empirical force field parametrized for macromolecular simulations to accurately locate the binding site of 3 . because most molecular mechanics ( mm ) simulation programs lack parameters for metal complexes , the x - ray data for 13 ( nine independent experimental structures ) were first used to develop a force field for the au macrocycles . we augmented the sp4 force field ( within ammp ) with parameters to model 15 and related au chelates . the new force field rivals density functional theory ( dft ) in structural accuracy but at a fraction of the computational cost ( table s2 ; figures s5 and s6 ) . we then devised a conformational search strategy ( figures 7a and s7 ) to determine the energetically preferred intercalation point for 3 along the length of the 22-bp dna duplex typically employed for x - ray structure determinations of top1 with its dna substrate ( figure 7d ) . ( in this respect it is noteworthy that classical force fields perform surprisingly well when compared with computationally expensive high - level quantum chemical simulations for calculations of both the structure of dna and , importantly , base - pair stacking energies . ) ( a ) conformational search strategy with a 22-bp dna duplex sequence [ part ( d ) ] favored by top1 and a modified sp4 force field for macromolecular simulations . a macrocycle such as 3 may intercalate between an adjacent base pair in one of four energetically distinct ways . the search proceeds in a manner akin to stepping down the rungs of a ladder . the compound intercalates at the 5-ta-3 site ( t10-a11 ) via major groove entry . ( c ) structure of the next - lowest energy conformation ( minor groove t10-a11 intercalation adduct ) of dna3 . the higher energy of this adduct reflects , in part , the non - planar conformation for the dna - bound au macrocycle relative to the global minimum . ( d ) graph of thermodynamic stability vs base pair ( left ) for the 22-bp dna duplex . ghelix is the empirical free energy penalty for strand separation at the specified base pair . right : graph of the relative energies of all simulated dna3 intercalation adducts vs ghelix . the best - fit straight line for the major groove adducts is given by : erel = 154(6 ) ( ghelix ) the lowest - energy conformation ( figure 7b , table s5 ) has 3 intercalated at a 5-ta-3 site ( t10-a11 ) via the major groove . the butyl chain of the macrocycle juts out into the major groove , oriented downstream ( 5 to 3 ) . the next lowest - energy conformation ( erel = 45.6 kj mol , figure 7c ) has 3 intercalated via the minor groove ( butyl chain oriented downstream ) at the same dinucleotide pair ( t10-a11 ) . minor groove intercalation yields a less stable non - covalent adduct than major groove intercalation at a ta step for several reasons , as depicted in figure 8 ( and figures s46 and s47 ) and enumerated at this juncture . ( 1 ) in contrast to the major groove intercalation adduct where both pyrrole rings and parts of the quinoxaline ring of 3 are involved in stacking with a and t , only the edges of the quinoxaline ring of 3 interact with these bases in the minor groove intercalation adduct . ( 2 ) the extent of insertion of the macrocycle into the intrahelical space is markedly less in the minor groove intercalation adduct . ( 3 ) the au macrocycle is forced to adopt a somewhat more distorted ( non - planar ) conformation in the minor groove intercalation adduct , which leads to a 5.58 kj mol increase in the torsional strain energy relative to the conformer that best - fits the binding pocket by major groove entry . ( the total energy difference between the two au macrocycle conformations is 4.94 kj mol , with the conformer intercalated via major groove entry lowest in energy . ) ( 4 ) electrostatic interactions between the au ion and heteroatoms ( o , n ) of either a or t in the minor groove adduct are absent ( figure 8b ) . significantly , when embedded within the t10-a11 binding pocket the lowest - energy major groove intercalation adduct of 3 exhibits a short auo contact ( 3.13 ) involving the nearest carbonyl oxygen of t10 and the au ion . this electrostatic interaction ( primarily an ion dipole attraction ) evidently makes a vital contribution to the overall stability of the dna3 non - covalent complex . perhaps most significantly , the conformational search gives the base pair specificity of 3 when the unique intercalation adducts are ordered from lowest energy ( highest affinity ) to highest energy ( lowest affinity):where superscripts ( j ) and ( i ) are major and minor groove insertions , respectively . independent verification of the above prediction that the intercalation site for 3 ( t10-a11 ) precisely matches top1 s target site for strand scission is mandatory . the experimental thermodynamic stabilities of the 10 possible nearest - neighbor interactions ( e.g. , aa / tt , at / ta , etc . ) present in a dna duplex are known and underpin the empirical algorithm used to calculate the free energy penalty for helix unwinding ( ghelix ) by the program web - thermodyn . we used this method with a sliding window of two adjacent nucleotides to analyze the 22-bp dna duplex of interest ( figure 7d ) . as shown by the bar graph , the stability of the adjacent nucleotide pairs follows the order : ta < at < ac < ag ct < aa = tt < ga . the free energy penalty for separating the nucleotide pair 5-ta-3 is thus the smallest ( < 1 kj mol ) . the fact that top1 has evolved to effect single - strand scission of duplex dna at a 5-ta-3 site , a thermodynamic weak point in the double helix , is noteworthy . it is also clear that a metallointercalator such as 3 similarly targets a 5-ta-3 site in duplex dna and that because 3 and top1 share the same dna target ( substrate ) , compound 3 will necessarily function as a type 1 unconventional catalytic inhibitor of the enzyme . the scatter graph ( figure 7d ) compares the relative energies of the dna3 adducts with ghelix for dinucleotide pair separation . a good linear relationship exists for adducts formed by binding / entry of the intercalator via the major groove ( but not the minor groove ) . clearly , the stability order ( base pair specificity ) for the major groove dna intercalation adducts of 3 parallels the thermodynamic stability of the dinucleotide pairs along the 22-bp sequence because the intercalator binds via the major groove and has to part the bases , thereby locally unwinding the helix at the intercalation point . both major and minor groove intercalation of 3 at the energetically favored t10-a11 step in the 22-bp duplex lead to a number of characteristic conformational perturbations of the dna . we used the program w3dna to quantify these effects at the level of individual steps ( i.e. , complementary base pairs ) using the ammp - calculated structure of the intercalator - free 22-bp dna duplex ( shown in figure 7a ) as a conformational reference . figure 9 graphically depicts the most significant of the metallointercalator - induced conformational perturbations . relative to the reference b - form 22-bp duplex , which has a mean propeller twist , , of 11.9(2) , the binding of 3 by either major or minor groove intercalation at the t10-a11 step shifts the propeller twist from the range 30 to 15 to between 5 and 0 immediately after the intercalation point , after which returns to ca . the reduction in propeller twist reflects the more regular , coplanar arrangement of the dna bases which come into direct contact with the aromatic intercalator through -stacking ; the local ordering of the bases evidently extends from a11 to g12 , but no further . figure 9b highlights the marked buckling of the base pairs induced by intercalation of 3 at the t10-a11 step through either the major or the minor groove . the mean buckling parameter , , for the reference b - form 22-bp duplex measures 0(3) , as expected for an unperturbed b - form dna conformation . the intercalation of 3 , in contrast , leads to substantial buckling of the base pairs up- and downstream of the intercalation point with averaging 3(8) over the full 22-bp duplex for both intercalation adducts . + 12 immediately downstream of the t10-a11 step is pronounced ; such a conformational perturbation has been previously noted for the structure of adriamycin - intercalated dna . the intercalation of 3 at the t10-a11 step is further characterized by reverse opening ( ) of the 10-ta and 11-at base pairs ( max = + 10.1 for the 10-ta pair of the minor groove adduct ) relative to the reference b - form 22-bp duplex for which = 10(1) ( figure 9c ) . finally , as expected the rise between adjacent bases in the stack , z , increases at the intercalation point because the intercalator assumes the position of a hydrogen - bonded base pair in the stack . this is depicted graphically in figure 9c where the 11-at pair clearly exhibits z values of 7.2 and 7.0 for the major and minor groove intercalation adducts , respectively . the calculated z values are consistent with the experimental values for base rise that accompany the presence of a dna - bound intercalator ( ca . ( a ) view ( roughly perpendicular to the aun4 plane ) of the ammp / sp4-simulated structure of the binding pocket ( t10-a11 ) of the lowest - energy intercalation adduct of 3 with the 22-bp dna duplex given in figure 7d . the -stacking between the bases and the pyrrole and quinoxaline rings of 3 is highlighted along with the juxtaposition of a t10 carbonyl oxygen atom ( red sphere ) and the au ion ( yellow sphere ) of the intercalator . ( b ) top view of the minor groove intercalation adduct highlighting partial intercalation of the au macrocycle and the practically negligible overlap of the pyrrole rings with the bases of the binding pocket . this view explains why the minor groove t10-a11 intercalation adduct is 45.6 kj mol higher in energy than the lowest - energy conformation . in both structures , the dna is rendered as a stick model in shades of gray and lilac , h atoms are omitted for clarity , and 3 is shown as a ball and stick model ( c , green ; n , blue ; au , gold ) . the two inset illustrations show edge - on projections viewed from the major to minor groove of the upper structure in both cases ( left image , van der waals radii including h atoms ; right image , ball and cylinder model with h atoms omitted for clarity ) . analysis of key conformational parameters for the two lowest - energy dna3 adducts formed by major and minor groove intercalation of the au macrocycle at the t10-a11 step of the 22-bp duplex depicted in figure 7 . ( a ) graph of propeller twist ( ) as a function of base pair index for bases 419 . ( b ) graph of buckling angle ( ) as a function of base pair index for bases 419 . ( c ) bar graphs of base - pair opening ( , left ) and rise ( z , right ) for selected base pairs close to the intercalation point . although a range of base pair specificities seem to exist for metallointercalators and are probably ligand and metal ion dependent , the recently determined x - ray structure of dna - bound -[ru(phen)2(dppz ) ] , where phen is 1,10-phananthroline , showed that the most symmetric ( of many ) intercalative binding modes for this cationic minor groove metallointercalator involved a 5-ta-3 site in a palindromic duplex dna sequence that was characterized by deep intromission of the dppz ligand into the dna intrahelical space in a manner akin to the major groove intercalation adduct calculated for 3 ( figure 8a ) . ( note that the quinoxaline ring of 3 is essentially two - thirds of the phenazine ring system in dppz , so the intercalative components of the two ligand systems are to a substantial extent comparable . ) interestingly , -[ru(phen)2(dppz ) ] did not intercalate at a 5-at-3 site in an analogous palindromic duplex dna sequence . despite the different trajectories preferred for dna intercalation by 3 and -[ru(phen)2(dppz ) ] , the foregoing experimental observation of ta over at specificity parallels the thermodynamic dna base pair specificity determined here for 3 using macromolecular simulations . note that the coordinatively saturated metal ion in -[ru(phen)2(dppz ) ] does not interact with any dna bases and that the phen ligands seemingly direct the trajectory of intercalation ( minor groove to major groove ) through non - covalent interactions with the minor groove nucleotides at the binding site . interestingly , the x - ray structure of dna - bound -[ru(bpy)2(dppz ) ] , where bpy is 2,2-bipyridine , exhibits similar minor groove intercalation , but with a rather different dinucleotide pair specificity to the former ru complex ( binding at central and terminal 5-at-3 and 5-cg-3 steps , respectively , being favored ) . since the two ru complexes under discussion differ only in their ancillary ligand pairs ( phen vs bpy ) , the non - innocence of the co - ligands in directing ( at least partly ) the site of dppz intercalation is highlighted . the intercalation mode calculated here for 3 is therefore distinct , more closely parallels that observed for major groove ta intercalation by -[rh(bpy)2(chrysi ) ] ( where chrysi = chrysene-5,6-quinone diimine ) , and evidently reflects the fact that the au ion is square planar and housed within a macrocycle with practically no steric bulk orthogonal to the mean plane of the quinoxaline and pyrrole ring systems . figure 8a gives a detailed view of the dinucleotide binding site of the lowest - energy structure of dna3 . as noted above , in addition to stacking interactions of adenine and thymine with the pyrrole rings of 3 , a significant 3.13- oau contact involving a carbonyl oxygen atom of t10 and the au ion of 3 indicates that the binding pocket interacts electrostatically with the metal ion . the simulations therefore explain why au is required for dna intercalation by the compounds . that 3 explicitly targets a ta rather than an at site is also apparent . specifically , if the order of the bases in the duplex is mutated to a10-t11 in the simulation , formation of the key thymine oau contact is obviated ( figure s48 ) . this a10-t11 intercalation adduct is , furthermore , 82.0 kj mol higher in energy than the global minimum energy structure ( t10-a11 intercalation via the major groove ) . if 3 binds at top1 s t10-a11 target site , can the enzyme still recognize its substrate and form a covalent cleavage complex ? we answered these questions by simulating the structure of wttop1 non - covalently bound to dna3 ( t10-a11 intercalation ) . from figure 10a , b , the quinoxaline ring of 3 protrudes into the minor groove and sterically blocks the enzyme s probe residue r364 from hydrogen bonding to n3 of g12 . furthermore , as shown by the overlay of the simulated structure of top1dna3 with the x - ray structure of the top1 y723f mutant ( mtop1 ) bound to the 22-bp dna duplex ( figure 10b ) , intercalation of 3 engenders partial unwinding of the dna helix downstream of the intercalation point . the strand shift measured by the displacement of g12 is 3.53 , consistent with -stacked 3 assuming the position normally occupied by a nucleobase ( a11 ) and the magnitude of the base rise ( z = 7.2 ; figure 9c ) . importantly , the scissile strand s t10-a11 phosphodiester link shifts 1.52 downstream , potentially thwarting attainment of the transition state for covalent tyr o p bond formation . our macromolecular simulations clearly predict that top1 will neither bind its dna substrate nor form a cleavage complex in the presence of 3 . dna intercalation by 3 , the ensuing structural perturbations , and their impact on dna binding by top1 . ( a ) lowest - energy simulated structure of the wild type top1dna3 ternary complex illustrating steric displacement of arg-364 and asp-533 by the quinoxaline ring of 3 protruding out the minor groove . these probe residues of the enzyme are key to dna substrate recognition . ( b ) overlay of the simulated structure displayed in part ( a ) with the x - ray structure of the y723f top1 mutant ( mtop1 ) bound to dna ( pdb code : pdb1a36 ) . ( c ) dna binding by mtop1 in the presence and absence of 3 determined by spr ( surface plasmon resonance ; 37 c , 41 l min flow rate , ph 7.4 , 10% dmso ) . the on - chip duplex dna sequence is illustrated beneath the graph ; the enzyme s ta dinucleotide target is highlighted . the biotinylated 20-bp duplex was anchored to avidin bound to the sensor chip surface . a conceptual illustration of the relevant equilibrium is given above the graph of spr response functions . the foregoing predictions were experimentally verified by determining whether or not mtop1 , a catalytically inactive mutant analogue of top1 , binds to an oligonucleotide target fixed to a surface plasmon resonance ( spr ) chip in the presence and absence of 3 ( figure 10c ) . the mutant enzyme ( mtop1 ) contains a point mutation at the active site tyrosine ( y723f ) which does not appreciably alter overall protein structure or dna binding affinity but destroys the ability of the enzyme to engage dna in a cycle of cleavage and religation ( which would complicate our analysis of dna binding ) . in this experiment , the surface of the spr chip was derivatized with a biotinylated 20-bp dna duplex before passing a solution of mtop1 over the chip . an spr response function commensurate with protein uptake ( 40100 s ) followed by saturation ( 100160 s ) and desorption ( > 160 s ) was obtained . after flushing the chip with buffer , a solution of mtop1 and 3 ( 50 nm ) was passed over the chip as before . no spr response was detected ( baseline signal in figure 10c ) , consistent with fast uptake of 3 by the dna and inhibition of enzyme binding by the immobilized dna3 intercalation adduct . finally , we garnered experimental proof that 3 binds to the ta sites of the 20-bp oligonucleotide . specifically , a solution of 3 passed over the dna - embellished spr chip afforded three stepwise association response functions prior to discrete desorption of 3 ( figure s50 ) . the data reflect the presence of the three 5-ta-3 sites along the synthetic 20-bp dna duplex and the fact that each has a unique microscopic affinity constant for 3 . the macroscopic kd values for 1 and 3 ( table s4 and figure s53 ) were 2.8 and 3.4 m , respectively , broadly in accord with their ctdna affinity constants ( figure 3a ) . importantly , 3 did not bind to mtop1 ( figure s52 ) , indicating that the enzyme is neither a primary nor a secondary target for the compound . based on the above spr data , dna binding experiments , and enzyme targeting assays , the lead compound of this study may be confidently assigned as an unconventional type 1 catalytic inhibitor of top1 . going forward , it will be interesting to ascertain whether the in vitro mechanism of action for 3 applies in vivo ( i.e. , in a chromatin setting ) . although such assays exist for ifps of top1 , non - emissive top1 cics such as 15 , which are unsuitable for confocal microscopy , currently present several unmet challenges regarding detection of endogenous top1 inhibition within cell nuclei . in summary , we have synthesized and characterized a new class of nominally planar cationic au macrocycles that incorporate two pyrrole - imine units linked to a quinoxaline moiety on one side and an alkyl chain bridge on the opposite side . from inception , physical measurements of dna binding by the compounds indicate that they are intercalators with high affinity constants ( ka > 10 m bp ) for ctdna that correlate interdependently with the lipophilicity of the salt and the steric bulk of the alkyl chain bridge within the macrocycle . hierarchical cluster analysis of nci-60 cytotoxicity data for the most active compound ( salt 3 ) indicated that 3 correlates most closely with the topoisomerase ib ( top1 ) poison camptothecin . several topical enzyme inhibition assays were used to prove that 3 is a catalytic inhibitor ( and not a poison ) of top1 . since catalytic inhibition of human topoisomerase ii ( top2 ) by 3 was 2 orders of magnitude weaker than its inhibition of top1 , compound 3 is a type i - specific agent . new mm parameters were developed for the sp4 force field for macromolecular simulations from the nine independent x - ray structures of the au macrocycles determined herein for parametrization . a conformational search strategy was devised to locate the lowest energy intercalation site ( adjacent nucleobase pair ) within a 22-bp dna duplex commonly used as a top1 substrate . the simulations showed that 3 intercalates dna at the enzyme s 5-ta-3 dinucleotide target sequence via major groove entry ( the minor groove adduct being > 45 kj mol higher in energy ) and that a crucial auo electrostatic interaction accounts for the observed base pair specificity . macromolecular simulations of a ternary non - covalent 3dnatop1 complex suggested that the molecular mechanism of action of dna - bound 3 is to block substrate recognition by the enzyme through steric repulsion . surface plasmon resonance studies confirmed ( 1 ) that top1 fails to bind its dna substrate in the presence of 3 , ( 2 ) that 3 does not bind to top1 itself , and ( 3 ) that the base specificity of 3 deduced by the macromolecular simulations ( ta ) is correct . the overarching conclusion of this multifaceted study is that the most cytotoxic au macrocycle , lead compound 3 , is an unconventional type 1 catalytic inhibitor of human top1 .

topoisomerase ib ( top1 ) is a key eukaryotic nuclear enzyme that regulates the topology of dna during replication and gene transcription . anticancer drugs that block top1 are either well - characterized interfacial poisons or lesser - known catalytic inhibitor compounds . here we describe a new class of cytotoxic redox - stable cationic au3 + macrocycles which , through hierarchical cluster analysis of cytotoxicity data for the lead compound , 3 , were identified as either poisons or inhibitors of top1 . two pivotal enzyme inhibition assays prove that the compounds are true catalytic inhibitors of top1 . inhibition of human topoisomerase ii ( top2 ) by 3 was 2 orders of magnitude weaker than its inhibition of top1 , confirming that 3 is a type i - specific catalytic inhibitor . importantly , au3 + is essential for both dna intercalation and enzyme inhibition . macromolecular simulations show that 3 intercalates directly at the 5-ta-3 dinucleotide sequence targeted by top1 via crucial electrostatic interactions , which include stacking and an auo contact involving a thymine carbonyl group , resolving the ambiguity of conventional ( drug binds protein ) vs unconventional ( drug binds substrate ) catalytic inhibition of the enzyme . surface plasmon resonance studies confirm the molecular mechanism of action elucidated by the simulations .

Introduction Results and Discussion Conclusions

PMC5102163

the physics involved ranges from subnanosecond submillimeter electron avalanches governed by atomic and molecular ionization and attachment cross sections to centimeterscale propagating space charge waves ( streamers ) to microsecondscale meterlong channels of ionized gas described by nonequilibrium plasma and electrodynamic behavior . on a larger scale , spark behavior in nature ranges from 100 km lightning channels that last seconds and span conditions from the upper reaches of a thundercloud to the microsecondscale processes by which such a channel attaches to the ground . in such a diverse range of behavior one can find many puzzling phenomena . the process by which a discharge initiates in the cloud is not understood nor is the process by which lightning channels ( leaders ) extend and branch . the extension process sometimes proceeds as a series of microsecondscale steps separated by 10 s of microseconds of relative quiescence . more puzzling , the steps seem to be immediately preceded by formation of a hot conductive channel displaced from the end of the existing channel that rapidly connects with the main channel , though the mechanism by which this space leader the transition from streamer to leader is the focus of much research as are the dynamics of the streamer itself and the role of energetic particles in the process . this role of energetic particles is especially interesting in the light of observations of xray production by lightning and the hypothesis that energetic particles produced by lightning contribute to the production of terrestrial gamma ray flashes . xrays as produced by lightning seem to occur coincident with stepwise extensions of the channel [ howard et al . , 2010 ; dwyer et al . , 2011 ] and have energies around a few 100 kev [ moore et al . , 2001 ; dwyer et al . , 2003 ; these xrays imply the existence of a much larger population of higherenergy electrons . while lowenergy electrons encounter high dynamic friction , relativistic electrons encounter much lower friction and can run away to very high energies when driven by electric fields , a fact first recognized by wilson and modeled in the context of electric fields near lightning channels in carlson [ 2009 , chapters 5 and 6 ] . seed energetic electrons necessary to initiate such a process can come from energetic background radiation ( i.e. , cosmic rays [ carlson et al . , 2008 ] ) , lowenergy electrons in local electric fields strong enough to overcome the maximum friction force [ e.g. , moss et al . , 2006 ; li et al . , 2009 ; chanrion and neubert , 2010 ] , or by feedback from prior generations of energetic electrons [ dwyer , 2003 , 2007 ] . regardless , electric fields may drive avalanche growth of populations of such runaway electrons[gurevich et al . unfortunately , the relative importance of these processes for phenomena like xray production by lightning or terrestrial gamma ray flashes is not well understood . in the context of these puzzles , any additional information about the processes involved , laboratory studies can shed light on the detailed dynamics of leader extension , and while the energy scales are necessarily smaller ( of order 1 mv compared to the 10 s of megavolts of natural lightning ) , lab sparks even produce xrays . dwyer [ 2005a ] report the first detection of xrays associated with such sparks in a study of 14 discharges of 1.5 mv , with total energy deposited in a detector up to mev scale . variation of signals among xray detectors with different attenuators suggest xray energies from 30 kev to 150 kev piling up to produce the mevscale observations . report similar results with total energy deposited up to several mev with 1 mv discharge . ( elaborated in nguyen ) also report similar results for 0.881 mv sparks with a much larger data set and with a variety of detectors , attenuators , and positions of detector . nguyen et al . show intensity variations with highvoltage ( hv ) polarity and with distance between detector and spark gap , suggesting that such intensity variations could result from xray production by positive streamers from metal structures near the detectors . report a further study of 241 discharges of 1 mv with various polarities and gap distances and push the maximum energy deposited in a single detector up to 50 mev with varied attenuators providing evidence for individual photon energies exceeding 300 kev and statistical evidence for an average photon energy at most 230 kev . dwyer et al . also report collimator experiments supporting the production of xrays by a diffuse source in the gap between the electrodes and that bursts of xrays produced late in the discharge come from elsewhere . march and montany report that hv pulses with rapid risetime tend to produce more xrays , and march and montany examines the effect of electrode geometry . kochkin et al . , kochkin et al . , and kochkin et al . add nanosecondresolution photography as a useful tool , mapping the streamer clouds produced by positive [ kochkin et al . , 2012 ] and negative [ kochkin et al . , 2015 ] hv discharges . in such experiments the hv risetime is much longer than the time for streamers to propagate across the gap , and in experiments with negative high voltage the negative streamers tend to appear and grow in bursts [ see , e.g. , kochkin et al . , 2014 , figure 6 ] . demonstrate for negative hv that the xrays tend to be emitted during these bursts of streamer development but are emitted on a much shorter timescale than the burst , supporting the suggestion that the large and transient electric fields that result from interaction of negative and positive streamer fronts may play a role in energetic electron and thus xray production . [ 2012 , 2015 ] also describe that attenuator experiments they claim are consistent with 200 kev characteristic xray energy . these estimates are based simply on comparison of registration rate ( the fraction of sparks for which xrays are observed ) for various attenuators with the expected attenuation in number of photons incident on the detector , assuming all sparks emit the same number of photons and that pileup does not affect their observations , though they acknowledge that pileup has a significant effect . together , these studies provide a reasonably complete picture of xray emissions from sparks : free electrons are pushed to overcome friction in the highfield region ahead of a negative streamer ( possibly briefly enhanced by a nearby positive streamer ) , gain at least enough energy to run away in the lower fields surrounding the streamer , then undergo bremsstrahlung in the surrounding air to emit xrays . these intense streamer fields exist only as the discharge develops and are unlikely to exist during the discharge itself , though the intense electrodynamic environment during discharge may drive discharges elsewhere in the lab that also produce detectable xrays . average energy , the properties of the photon spectrum are unknown and difficult to judge due to pileup , and the fact that some sparks produce copious xrays while others produce no measurable xrays is not addressed . the present paper attempts to shed light on these topics by examining the energies measured in xray detectors placed near a 1 m spark gap at the technical university of eindhoven during 921 shots as described in section 2 . these shots give us the opportunity to statistically analyze the distributions of spark xray fluence , number of photons detected by multiple detectors , and photon energy . these distributions are described in section 3 and constrained by comparison to observations in section 4 . the experiment was conducted in the highvoltage lab at the technical university of eindhoven with a haefly 2 mv marx generator configured to produce 1 mv pulses with 1 s risetime . all tests were carried out with a 1 m pointpoint gap with negative hv polarity . the spark voltage , ground electrode current , and highvoltage electrode current were recorded by lecroy fourchannel storage oscilloscopes configured to record 2 s of data at 10 ghz sample frequency when triggered by the marx generator , as were signals direct from the output of photomultiplier tubes monitoring a variety of energetic radiation detectors . the radiation detectors used in the experiment included scintillating plastic optical fibers placed at a variety of locations around the spark to monitor energetic electrons . analysis of these data has been presented [ ostgaard et al . , 2014 ] and will be published separately . due to the need to run many shots with the scintillating fiber detectors at various locations , a total of 950 shots were carried out from which we recorded usable data for 921 . during all shots , two additional xray detectors were running , providing a wealth of data about the photon population . these two detectors are each composed of a 1.5 inch long 1.5 inch diameter labr3(ce ) scintillator monitored by a photomultiplier tube ( pmt ) . these detectors are placed next to each other ( separation 10 cm ) in an electromagnetic compatibility ( emc ) cabinet to shield the detectors from the electromagnetic noise produced by the spark . the location of the detectors relative to the spark corresponds to location h as shown in figure 1 of kochkin et al . . the scintillator material is separated from the spark by roughly 0.5 mm of aluminum in the scintillator housing and emc cabinet wall and approximately 2 m of air . the signals as recorded by the oscilloscopes are in volts and here have been converted to mev by use of cs137 and co60 gamma ray emitters as sources of known energy . the lowest energy detectable by this setup is 20 kev , comparable to the minimum photon energies transmitted through the 0.5 mm aluminum shielding , while the maximum depends on the settings of the oscilloscope . signal from detector 1 clips at just above 5 mev , while detector 2 clips at just above 3.5 mev . the highvoltage trace shown is very consistent from one spark to the next , as is the highvoltage electrode current . the pulselike features on the highvoltage electrode current occur when bursts of corona and streamer activity carry charge away from the electrode . see kochkin et al . for a detailed discussion of such processes as seen in highspeed camera imagery . ( top left ) the voltage of the highvoltage electrode with respect to the ground electrode . the pulses seen in the oscilloscope traces indicate deposition of energy in the scintillator , but a single pulse from the pmts may be produced by multiple xray photons entering the scintillator . these photons may arrive at slightly different times and produce a visibly altered pulse shape , but typically no time structure is visible . the photon spectrum is therefore not directly measurable , and pulse pileup must be treated statistically . to compile data for such statistics , we search through the data for pulses . to search , we first smooth the data by lowpass filtering once pulses are identified , we collect a variety of data including pulse time , height , integral , and duration . pulse integral is a more robust measure of deposited energy in case multiple pulses arrive near simultaneously , so our analysis here reports energy as determined by pulse integral . while pulse integral is somewhat resistant to saturation when oscilloscope signals clip , the energy of a saturated pulse can not reliably be determined , so we enforce a maximum energy for each detector corresponding to the energy of the largest unclipped pulse . in cases when multiple pulses are observed in a single detector , we add their energies together to better capture variation from one spark to the next , though note that the majority of sparks have zero or only one burst so this combination of bursts only affects a small fraction of our data . the data set we consider here is therefore a set of pairs of numbers , each pair associated with a single spark and each number with the pair giving the total energy deposited in a detector during the given spark . a scatterplot showing the energies deposited in each detector in each shot is shown in figure 2 . most sparks ( 57% ) produce no detectable signals in either detector , indicating less than 20 kev deposited , while approximately 3% of sparks saturate both detectors , indicating at least 35 mev of energy deposited . each point represents a spark , where e 1 was deposited in detector 1 , while e 2 was deposited in detector 2 . the dotted line shows e 1 = e 2 . at e 1 = e 2 = 0 , 523 points overlap , and 20 points the points clearly cluster in the lower left , but a significant number of points appear in the middle and upper regions of the plot . attempting to explain this distribution most simply , one might assume all sparks are identical and that all photon energies are equal and treat the observed distribution as solely due to poisson statistical fluctuations in the number of photons observed . in this case , the high number of points in the lower left ( 57% of events undetected ) implies a poisson mean of 0.562 . with this mean , observation of events that saturated both detectors is then incredibly unlikely as deposition of more than 8 mev ( 3 mev in one and 5 mev in the other ) requires at least 40 photons for the mean energy 200 kev consistent with the results quoted above . observation of at least 40 photons from a poisson distribution with mean 0.562 has a probability around 10 , much less than the 3% of observed sparks that saturate both detectors . adding the complication of a photon energy distribution is logical , but does not help enough , since the maximum photon energy must be less than 1 mev as the photons will be much less energetic than the 1 mv maximum spark voltage . assuming the same poisson mean as before and attempting to explain the events that saturated both detectors as due to an extreme fluctuation in photon energy such that all photons carried 1 mev of energy means that we must have at least eight photons instead of at least 40 , but the poisson probability of observing at least eight photons given a mean of 0.562 is roughly 10 , still much smaller than the 3% observed . clearly , there must be a significant variability in spark xray fluence at the location of the detectors from one spark to the next . this variability could come from intrinsic variability in spark xray luminosity or it could come from some variability in the geometry if xray emissions are not isotropic . regardless , a full explanation of the distribution of points in figure 2 must include the effect of the distribution of spark xray fluence , the distribution of numbers of photons detected by each detector ( given the xray fluence ) , and the distribution of photon energies . these distributions and their properties are the focus of this paper . in attempting to build a statistical model of the distribution of points in figure 2 , we need to know the form of the distributions of physical properties relevant to the point locations . there are three main distributions at work : the distribution of spark xray fluence , the distribution of numbers of photons incident on each detector ( given spark fluence ) , and the distribution of photon energies . the distribution of spark fluence is determined in principle by the processes at work in electron acceleration . one spark has an intrinsically higher luminosity than another by having more energetic electrons , perhaps because random streamer branching events happened to lead to more negative streamers or perhaps fewer but more intense or more rapidly growing negative streamers or perhaps more interactions between negative streamers and positive streamers . fluence variability may also result from anisotropy in the directionality of xray emissions : perhaps all sparks emit many xrays , but the emissions are not always beamed toward the detectors . these processes may be chaotic or perhaps intrinsically random , but either way there will be some variability in xray fluence . to represent this variability , we treat the xray fluence as the expected value of the number of photons hitting a detector , , and assume some probability distribution for the occurrence of various values of . as there is yet no theoretical expectation for the form of this distribution , we must make some assumption . examining the clustering of points in the lower left of figure 2 , we expect lower fluences to be more common than higher fluences , but the existence of points in the upper right suggests that the tail of the distribution to high fluences is quite strong . to represent distributions that follow this general trend of decreasing with a potentially long tail , we assume the distribution for is a power law with index : ( 1)p()=(+1)max+1min+1 where min and max are lower and upper limits on necessary to ensure that the distribution is normalizable for 1 and the leading constant ensures normalization . by examining the clustering in the lower left in figure 2 , we expect < 0 , but the number of points saturating both detectors suggests that is not too negative and that the distribution of is in some sense quite hard . given the fluence of the spark ( i.e. , given ) , the number of photons hitting a detector , n , should be poisson distributed with mean . normalized , ( 2)p(n)=nen ! this assumes that there is a very large number of photons produced by the spark , each with a very small probability of hitting the detector , and that the two detectors have the same probability of catching each photon . this implicitly assumes that xray emissions from the spark are uniform on the scale of the 10 cm separation of the detectors , but this is reasonable as the detectors are roughly 2 m from the spark and bremsstrahlung from electrons with only a few hundred kev of energy is not strongly beamed . finally , given a number of xrays hitting each detector , one can calculate the distribution of deposited energy by applying a distribution of xray energies . this unfortunately is unknown as we do not know the distribution of electron energies responsible for the bremsstrahlung emissions [ chanrion and neubert , 2010 ] . figure 2 presents calculations of energy distributions of free electrons as produced in constant electric fields that appear linear on a semi log plot ( inset ) and that show the effects of an exponential cutoff up to the threshold of runaway electron behavior . the resulting photon distribution will share some of these characteristics , but in principle connecting the photon and electron distributions requires detailed treatment of bremsstrahlung , a topic beyond the scope of the current work . as such , we simply assume that the photon energy e is also exponentially distributed with mean photon energy : ( 3)p(e)=ee/ where the division by ensures normalization for 0<e<. note that for simplicity we are assuming that the photon energy distribution does not depend on xray fluence . it is reasonable to expect that depends on somewhat , but including this dependence would require still more assumptions and additional free parameters . assuming an exponential photon energy distribution is especially convenient since the distribution of energies deposited in a detector e , a sum of the energies of n photons ( e=i=1nei ) will be distributed as the sum of n independent exponentially distributed random variables . the distribution of such a sum of exponential random variables can be expressed in closed form and is known as the erlang distribution : ( 4)pn,(e)=en1ee/n(n1 ) ! note , however , that if n = 0 , no photons strike the detector , the energy deposited is exactly zero , and p n = 0,(e ) = (e ) , a dirac delta function , instead of the continuous distribution above . if the fluence of the spark ( ) and the number of xrays hitting each detector ( n 1 , n 2 ) were known , the joint distribution of energy depositions would simply be given by a product of two erlang distributions . unfortunately , the fluence of the spark and the number of xrays hitting each detector are not known . as such , we must compute the marginal distribution of observed energies by integrating ( i.e. , taking an weighted average ) over the possible values of the unknown quantities , weighted by the probability distributions associated with those quantities . for one detector , ( 5)p,(e)=minmaxdp()n=0p(n)pn,(e ) and for the joint distribution of simultaneous observations with two detectors , ( 6)p,(e1,e2)=minmaxdp()n1=0n2=0p(n1)pn1,(e1)p(n2)pn2,(e2 ) where the integral over covers the range of possible spark xray fluence and the sums over n or n 1 and n 2 cover the possible numbers of photons observed by each detector . the resulting joint probability distribution of e 1 and e 2 should be able to reproduce the observations in figure 2 there is a finite probability that both detectors observe exactly zero energy , a discrete probability represented by the n 1 = n 2 = 0 term in the sums for which pn1, and pn2, become delta functions as described above . likewise , the n 1 = 0 , n 2>0 , and n 2=0 , n 1>0 terms in the probability distribution contain a single delta function . only the terms for which both n 1 and n 2 are greater than zero lack delta function divergence behavior . these delta functions make the probability distribution difficult to work with directly but are capable of representing the clustering of points at the origin and along the axes in figure 2 . these probability distributions form the basis of our model , and we seek to infer the parameters , , min , and max by comparison of our model to data . comparison of a probability distribution to data can be done in many ways , for example , the kolmogorovsmirnov test or the andersondarling test . such tests work on the basis of the cumulative distribution function ( cdf ) f , which for a probability distribution p(x ) in one dimension is defined as f(x)=xp()d. since here we work in two dimensions ( e 1,e 2 ) , we need a twodimensional analog of the cumulative distribution function . here we follow fasano and franceschini in taking our set of data points { p i } = { ( e 1i , e 2i ) } and for each point , assigning four cdflike values : fdati < < , fdati< , fdati < , and fdati , each giving the fraction of the observed data set in the corresponding quadrant relative to the data point in question . for example , fdati < < gives the fraction of data { p j } for which e 1j < e 1i and e 2j < e 2i . these { fdati } can be plotted if an order is assigned to the data points for use as an abscissa . here it is convenient to order each set of fdati separately such that fdati is monotonically increasing , i.e. , sort each set of fdati from smallest to largest and use its place in the resulting list as its abscissa . for example , the grey , curve starts at 0.03 since 3% of sparks saturate both detectors : the data point that has the fewest data points with energies greater than or equal to its energy must be one of those doubly saturated events , and thus , 3% of the data satisfies the , condition . moving to the right along the , curve , larger cdf values correspond to data points that are below and/or to the left of the upper right corner , i.e. , data points for which larger fractions of the data satisfy the , condition . the large jump from about 0.35 to 1.0 at cdf rank 400 occurs when incrementally increasing the fraction of the data set that satisfies the , condition requires looking at a point where e 1 = e 2 = 0 , which thus includes all other such points ( 57% of all data ) as well as points for which e 1 = 0 or e 2 = 0 . the cdf therefore jumps up to 1 since all of the data has e 10 and e 20 . the direction of the inequality is related to the shape of the distribution , with and < < related to the lower left / upper right balance and width while the < and < versions related to upper left / lower right balance and width . one can imagine other ways of constructing such cdfs , but this is a straightforward technique that as we will see shortly is quite effective . each horizontal point represents a point in the ( 2d ) data set , for which the vertical coordinate represents the fraction of the data set with energies related to the energies of the given data point by the inequalities associated with the given curve ( i.e. , the fdati described in the text ) . each curve represents a different inequality , and the data have been reordered for each curve to make the curve in question monotonically increasing . two sets of curves are shown , thick curves from the data and thin curves predicted given the best fit parameters discussed in the text , but the curves overlap so much any deviations are difficult to judge . such curves as in figure 3 can be predicted on the basis of the probability density ( equation ( 6 ) ) , assigning a predicted fpredi to each data point by integrating the expected distributions over the region relevant to the inequalities in question . for example , ( 7)fpredi<<=e1ide1e2ide2p,(e1,e2 ) similar calculations can be made for predicted fpredi < , fpredi< , fpredi by changing the limits on the integrals to go from the point in question to + as appropriate . regardless , the exponentials and powers in the integrand can be manipulated to convert the integrals here and in equation ( 6 ) into upper and lower incomplete gamma function evaluations ( e.g. , (k , x)=xxk1exdx ) , and the summations in equation ( 6 ) can be computed numerically and truncated without significant loss of accuracy . for a given , , min , and max , the result of this exercise is four sets of numbers ( { fpredi < < } , { fpredi < } , { fpredi< } , and { fpredi } ) that can be compared to the corresponding sets associated with the data ( { fdati < < } , { fdati < } , { fdati< } , and { fdati } ) . treating these numbers as analogous to the cumulative distribution function , the kolmogorovsmirnov test statistic would be the maximum deviation between any two corresponding f i . the kolmogorovsmirnov test is sometimes criticized as not very powerful [ e.g. , razali and wah , 2011 ] , so instead , we calculate an analog of the andersondarling test statistic : ( 8)s=i(fdati<<fpredi<<)2fpredi<<(1fpredi<<)+(fdati<fpredi<)2fpredi<(1fpredi<)+(fdati<fpredi<)2fpredi<(1fpredi<)+(fdatifpredi)2fpredi(1fpredi ) i.e. , the squared deviation between the predicted cdf and the observed cdf , weighted as in the andersondarling statistic , summed over all four cdf types ( inequalities ) , and summed over all points in the observed data set . some numerical problems arise when both numerator and denominator are exactly zero , i.e. , one of the fpredi is exactly 0 or 1 , as occurs , for example , for fpredi < < for a point , where e 1i = e 2i = 0 . since such points involve exact match between predicted and observed cdf and therefore should not contribute to the test statistic , we add a small factor ( 0.001 ) to the denominator of each term to ensure the division of zero by zero results in zero . this does not significantly affect the other terms in the test statistic or the overall results . we then fit our predicted distribution to the observed distribution minimizing s by varying the parameters of our calculated cdf with the downhill simplex algorithm ( nelder and mead as implemented in johnson ) . the optimal values are = 1.29 , = 86 kev , min = 0.022 , and max = 113 . since our multidimensional cdf and modified andersondarling test statistic are so far removed from their original application , we only use the statistic in judging the quality of the fit in the optimization process described above and make no attempt to apply the distributions typically associated with the andersondarling test statistic to assess the uncertainty in our fit results . instead , we judge uncertainty by bootstrap , repeating our fit process many times with alternative data sets produced from our original data by sampling with replacement . the fit results are each approximately normally distributed , with mean and standard deviation given as follows : = 1.29 0.04 , = 86 7 kev , min = 0.022 0.006 , and max = 110 25 . plotting these bootstrapped fit results , one parameter versus another , shows very little correlation between results , with the exception of and min which are negatively correlated : more negative is associated with higher min . this makes sense given that more negative is associated with more events with low xray fluence , so raising the minimum fluence is necessary to retain the balance between events with and without detectable signal . the cdfs computed from the best fit parameters are also shown in figure 3 as thin curves , but they overlap with the data so much that no systematic deviations are evident . a residuals plot showing the differences between the two sets of curves is shown in figure 4 . some deviations can be seen but are well within the typical size of the fluctuations due to random distribution of points in the data set . a positive deviation indicates a larger data cdf than computed from the best fit parameters . monte carlo simulations of data sets drawn from the distributions described above show no obvious deviations from the distribution of the data when plotted as in figure 2 . it is also worth noting that since our fitting procedure captures the joint distribution , it also captures the distribution within each detector separately . in this paper , we have not attempted to compensate for saturation to construct a true energy distribution , but kochkin et al . , using essentially the same experimental setup as used here , do present such a spectrum [ kochkin et al . , 2015 , figure 12 ] constructed by a sophisticated procedure of fitting pulse shapes to observed oscilloscope records . though the analysis in kochkin et al . includes the data set used here , the analysis procedures are completely different , and kochkin et al . results are presented for energy deposition per burst not per spark , but the fact that relatively few sparks have multiple bursts means that it is still useful to directly compare the results from kochkin et al . to equation ( 5 ) . evaluation of equation ( 5 ) with our fit result values of , , min , and max , normalized and overlaid with the data from kochkin et al . , is shown in figure 5 , demonstrating much better agreement than the single exponential distribution used in kochkin et al . . inferred a 200 kev characteristic burst energy ( which is asserted to be roughly equal to characteristic photon energy ) but showed an extremely poor fit at high burst energies that they attribute to pileup . properly accounting for pileup as we do here with the poisson distribution of observed counts ( p (n ) ) and for fluence variability with the assumption of a power law ( p ( ) ) gives us a much better fit with a much lower mean photon energy ( 86 kev ) , suggesting that pileup and fluence variation is essential to consider in interpretation of such data . comparison of the predicted total energy deposition spectrum from this paper with observations from figure 12 of kochkin et al . . the red squares show the observed spectrum , while the black curves show the predictions based on our distributions . the thick curve is the prediction , while the thin curves represent the n standard deviation expected for the spread in the data . as a final comparison to data , while we ran no attenuator experiments in the data set described above , kochkin et al . they report results for lead attenuators of varying thickness , determining the fraction of sparks that produce observable signals in a single detector by running 50 sparks with each attenuator . the distributions determined above can easily be used to construct results for attenuated scenarios for comparison with the observations in kochkin et al . by monte carlo simulation . first , we draw a random spark fluence , then draw a random number of photons from such a spark , then draw that many random photon energies from the photon energy spectrum , then attenuate those photons probabilistically by use of the mass attenuation coefficients from hubbell and seltzer , and then finally classify the event as containing detected xrays or not based on whether or not any photons made it through the lead shield . we repeat this procedure 10 times , compute the overall fraction of events with detected xrays , and repeat for each attenuator thickness . comparison with data requires an estimate of the uncertainty in the data , here estimated by calculating a 68% confidence interval based on the binomial distribution . sixtyeight percent confidence corresponds to a 1 error bar , and all but one of our predictions are consistent with the observations at this confidence level . the distributions determined in this paper are thus also consistent with attenuator experiments , though further attenuator observations would be useful to narrow the uncertainties in the observations . predictions based on the distributions determined in this papera the 68% confidence intervals correspond to 1 error bars and are calculated based on binomial statistics . to summarize , we have studied observations of xray emission by sparks and attempted to understand those observations by modeling the process as a combination of a distribution of spark fluence , poisson statistics , and an xray energy spectrum . these distributions successfully reproduce not only the data used here but also independent analysis of similar experiments on overall energy distribution and signal attenuation in kochkin et al . . the two main results are a welldefined photon mean energy and the distribution of overall spark fluence . the 86 7 kev mean photon energy determined above is consistent with most earlier estimates but somewhat lower than some . we feel this mean energy is quite reasonable given that electrons accelerated in the environment of a 1 mv spark realistically must have at most a few hundred kev of energy and bremsstrahlung photons produced must have a lower energy than that of the source electrons . as a crude estimate of electron energy , assume bremsstrahlung produced by the relatively low energy electrons here follows approximately the dn / de 1/e distribution seen for bremsstrahlung produced by highenergy electrons . this 1/e distribution spans the range from the energy of the electron as a maximum to a minimum energy at most 20 kev . using 20 kev as the minimum energy and requiring that the average photon energy is 86 kev require the electron energy to be roughly 200 kev , while using 5 kev as the minimum energy gives nearly 400 kev as the electron energy . this 200400 kev energy range is not inconsistent with the potential available for electron acceleration at the time of xray production as seen in figure 1 , but this is a crude estimate in need of refinement by modeling of electron energy distributions and the resulting bremsstrahlung before it can be taken very seriously . the exponential xray spectrum motivated by the expectation of an at least approximately exponential distribution in electron energies very closely reproduces the observed distributions , though is likely not the only such distribution to do so , and as noted above we have assumed that fluence ( ) and mean energy are independent which may or may not be true . while the average energy should be close to correct , the other distributions at work in this system complicate the analysis . the more important factor at work is the very broad distribution of observed spark xray fluence ( ) , which , as mentioned earlier , we assume captures both intrinsic variability in spark luminosity and variability in the spatial distribution of xray emissions . the results obtained above , which the distribution of is very hard ( power law index 1.29 ) and covers a broad range , from 0.02 photons to over 100 photons for the experiment conducted here , pose a challenge to the idea that the strong field near the head of a negative streamer is all that is required to accelerate electrons into the energy regime needed for 100 kev xray production . all sparks involve extensive streamer production [ see , e.g. , kochkin et al . , 2014 , figure 2 ] , so the number or distribution of streamer production can not directly explain the broad distribution of spark xray fluence . as discussed in kochkin et al . , the very short duration and appearance of multiple bursts of xray emissions suggests that xray emission occurs during some fast transient process like streamer collision , but it is not clear how streamer collision frequency would be distributed and whether such a distribution could reproduce the characteristics observed here . while we have not proved that xray fluence truly follows a power law , only that our assumption of a power law is consistent with our data , it is perhaps not unreasonable that a power law distribution might arise here in the context of dielectric breakdown since power laws arise in studies of systems that similarly approach breakdown and then collapse [ bak et al . , regardless of the true form of the distribution , we hope our analysis here is useful for evaluation of xray production mechanisms , which must explain the frequency of both dim and bright sparks in roughly the balance described here . as for the relative contributions of spark luminosity variation and nonuniform xray spatial distribution to the observed fluence variability the observed fluence variability should correspond roughly to the degree of spatial variability in xray emissions . one can set an upper limit on the spatial variability present in xray emissions in an experiment such as this by examining the directional distribution of bremsstrahlung from a unidirectional beam of 500 kev electrons . [ 1979 , figure 5 ] and khn and ebert [ 2014 , figure 7b ] . both figures present plots of the intensity of emissions versus angle relative to the direction of the electron beam , with tseng et al . showing a factor of around 100 difference from highest to lowest fluence , while khn and ebert show a factor of around 300 . that the best fit distribution constructed here requires a factor of max/ min = 100/0.02 = 5000 from highest to lowest fluence suggests that even this extremely aggressive assumption of unidirectional 500 kev electrons falls short of explaining the observed fluence variability , but this analysis is admittedly crude . a slightly less crude assessment can be made by monte carlo : first , draw a random beam direction uniformly from the highvoltage electrode in a hemisphere facing the ground electrode , then determine the angle from the center of that beam to the location of the detector and evaluate the relative fluence by extracting the distributions shown in tseng et al . and khn and ebert for a relatively high energy photon . though this ignores the xray energy dependence of the bremsstrahlung angular distribution , it should reasonably account for the degree of variability . repeat this procedure many times , compiling a histogram estimating the fluence distribution , then scale these histograms such that they are normalized and adjust the relative fluence to correspond to by shifting the relative fluence until the histograms correctly predict the fraction of events that would fall at intensities too low to be detected . histograms constructed in this manner are compared to the best fit power law in figure 6 . predicted fluence distributions assuming only spatial variability due to beaming of bremsstrahlung from unidirectional 500 kev electrons . the curve marked tseng is derived from figure 5 of tseng et al . , the similarity of slopes is striking , especially for the khn and ebert case ; but it is clear that fluence variability due to beaming can not fully account for the observations . in the context of the 86 kev average photon energy determined here , the maximum fluence of the bremsstrahlungderived cases , 10 , would result in 800 kev deposited on average , far from the 35 mev needed to saturate the detectors . put another way , if we attempt to account for the large fraction of lowfluence events simply as emissions beamed away from the detectors , the maximum fluence would then be too low to account for events that saturate the detectors . the scale of variability in the distributions derived from bremsstrahlung , even for the extreme case of unidirectional 500 kev electrons , is therefore too low to fully account for the variability observed , providing clear evidence for strong sparktospark variability . crudely comparing the spread of the khn and ebert curve to our power law in figure 6 , there is at least another order of magnitude variability in fluence that can not be explained as spatial variability and that is a very conservative estimate since in a more realistic scenario there will be directional dispersion of electrons , lower energy electrons , and possibly multiple beams , all of which lead to reductions in the spatial variability due to beaming alone , thus requiring even more sparktospark variation . we hope the results described above will provide a basis for comparison to results of other spark experiments to determine if the distributions of mean photon energy and spark xray fluence depend on the experimental setup . we further hope that theoretical work can find a basis for such distributions to confirm or refute our assumptions and that such theories can shed light on the process of runaway electron acceleration in sparks as suggested to be relevant to other electrical phenomena such as lightning or terrestrial gamma ray flashes .

abstractxray emission by sparks implies bremsstrahlung from a population of energetic electrons , but the details of this process remain a mystery . we present detailed statistical analysis of xray spectra detected by multiple detectors during sparks produced by 1 mv negative highvoltage pulses with 1 s risetime . with over 900 shots , we statistically analyze the signals , assuming that the distribution of spark xray fluence behaves as a power law and that the energy spectrum of xrays detectable after traversing 2 m of air and a thin aluminum shield is exponential . we then determine the parameters of those distributions by fitting cumulative distribution functions to the observations . the fit results match the observations very well if the mean of the exponential xray energy distribution is 86 7 kev and the spark xray fluence power law distribution has index 1.29 0.04 and spans at least 3 orders of magnitude in fluence .

Introduction Experimental Setup Statistical Model Comparison to Data Discussion

PMC3385362

long duration space flights expose human body to microgravity which affects the physiological health drastically . some of the physiological effects that result from human space flights are bone loss , muscle atrophy , cardiovascular deconditioning , altered sensory motor reactions , fluid redistribution / orthostatic intolerance , etc . in order to counteract these changes and increase astronaut performance in orbit , cardio vascular exercises as well as resistive exercises has been made a part of the international space station missions . it has been evident that extravehicular activity ( eva ) exposes the crew member to substantial physical stress . use of eva suits during mars analogue work , particularly those with helmets exhibiting poor ventilation , has been reported to cause increased exertion , expressed as heart rate and stress levels response , during short period of physical work . it is of utmost importance that the crew of aircraft that is to land on mars should possess adequate physical fitness in order to handle their work tasks and to be prepared for possible emergency situations . hence , the astronauts need to be physically fit and be capable of performing intense activities . it is known that heart rate and cortisol are one of the parameters that can be measured to know the stress levels.[57 ] in the present study we characterized the physiological cost of mars analogue eva work by outfitting crew members with a portable expired gas analysis system during training eva sessions . also , to find out the correlation between heart rates , stress and cortisol levels , the heart rates , cortisol and workload during eva . the six - crew members were selected from 100b ilewg euromoonmars during mdrs ( mars desert research station ) , utah , usa . the ages for the crew members ( n = 6 ) aged 20 - 26 [ 23.6 ( 2.4 ) ] years . the average and calcium intake of the crew members during mission was 2400 kcal / day ( range 2090 - 3200 kcal / day ) and 1267 mg / day ( 1130 - 1400 mg / day ) , respectively . dietary sodium and potassium intake were maintained at 98 ( 80 - 103 ) and 86 ( 75 - 120 ) mmol / day , respectively . water intake was ad libitum 1236 ( 68 ) ml / day ( 940 - 1590 ) ml / day . each crew members was outfitted for gas analysis during two eva excursions and two graded hill runs , each performed on a separate day . the eva excursions were for the purpose of training the crew for geological surveying , were between one and two hours in duration , and were performed while wearing the mars eva suit provided for at the mars desert research station ( mars society , usa ) . the hill runs consisted of continually running up and down a 200 m hill path ( 10% grade ) . peak was increased every two minutes at the encouragement of a principal investigator ( br ) and the participant was instructed to run until exhaustion . the peak 10 second mean value recorded over the two hill sessions was taken to represent vo2 peak ( peak oxygen uptake or maximal aerobic capacity ) . aerobic energy expenditure determined by measured oxygen consumption ( vo2 ) during eva sessions was compared relative to peak oxygen consumption ( vo2 peak ) recorded during graded hill runs performed on 14 separate days . the heart rates , cortisol , vo2 of the crew are measured before , during and after an eva . the workload of the crew is measured using nasa task load index ( tlx ) before and after the eva . expired oxygen and carbon dioxide were measured in a breath by breath manner by a cos med portable gas analysis system ( cosmed , italy ) . saliva samples were collected ( versi - sal , oasis , usa ) immediately before eva and after eva . the average vo2 peak recorded during the hill runs was 56.6 ( 2.67 ) ml / kg / min and peak vo2 reached during a given eva was 35.87 ( 0.78 ) ml / kg / min . data for heart rate showed the same trend as vo2 , with a maximal increase to 85% of peak [ figure 1 ] . the rating of subscale showed a significant increase in eva as compared to run [ figure 2 ] . also rating of subscale showed a significant increase during eva as compared to before and after . a good correlation between nasa tlx and salivary cortisol levels ( r = 0.68 , p < 0.001 ) . salivary cortisol levels were increased in both groups [ figure 3 ] , although significant increased of cortisol levels ( p < 0.05 ) more in eva as compared to hill running crew members . heart rates increased in both groups [ figure 4 ] , although significant increased of heart rates ( p < 0.01 ) more in eva as compared to hill running crew members . there was positive relation between the rise in salivary cortisol concentration and max vo2 ( r = 0.78 , r = 0.75 ; eva and hill running , respectively ) . there were good correlation between heart rate and cortisol levels ( r = 0.69 , r = 0.72 ; eva and hill running , respectively ) . peak heart rate achieved during hill runs and eav ( figure 1 showing that heart rate was significantly increased during eva task as compared to hill runs ) average task load index ( tlx ) during hill runs and eva ( figure 2 showing that average was significantly increased during eva task as compared to hill runs ) salivary cortisol levels during hill runs and eva ( figure 3 showing that salivary cortisol levels were significantly increased during eva task as compared to hill runs ) heart rate during hill runs and eva . ( showing that heart rate was significantly increased during eva task as compared to hill runs ) this pilot study showed that a typical mars analogue eva requires a sustained challenging physical effort . the rating of subscale showed a significant increase in eva as compared to hill run . it might be due to higher stress situation during eva task as compared to hill run which is supported by significantly increased of salivary cortisol levels and heart rates during eva task as compared to hill run . during exercise , when the max o2 consumption exceeds 60% an increase in the epinephrine and cortisol concentrations occurs . during stress condition vasopressin stimulates the release of corticotropin - relasing factor , which in turn leads to the release of acth ( adrenocorticotropic hormone ) . the highest value of cortisol was reported in aerobic capacity exercises . whereas anaerobic exercise for a brief period led to no changes whatsoever in the sporting subjects , with aerobic exercise an increase in the cortisol and acth hormone secretion was observed . a good correlation between nasa tlx and salivary cortisol levels the positive relation between the rise in salivary cortisol concentration and max vo2 was found . in the light of these findings a lack of accurate methods of vo2 max estimation by treadmill or cycle ergometer testing protocols and less number of subjects were main limitations of this study . notwithstanding of these limitations this investigation was able to observe authentic physiological phenomena in the unique environment of the mars analogue environment . continuation of aerobic fitness during the long trip to mars will be considerably more difficult . so , new training methods and medicinal therapy will have to be explored that can not only maintain aerobic fitness to the levels specified , but also address muscle atrophy and bone demineralization due to microgravity . these results suggest that salivary cortisol rhythms may be successfully employed for estimating circadian rhythms and related stress decrements in astronauts during space missions . we can use these salivary markers for mentoring of stress and workload parameters during selection of astronauts , during mission and after mission because being a noninvasive , cost - effective , less time consuming , easy to use and non - infectious . further study is required on large scale taken into account of limitations such as large sample size , no applicable of statistical analysis method due to small sample size , using standardized measuring methods , taking into account of more serum and salivary stress biomarkers , with and without helmet of this study and including other physiological and psychological parameters in mars analog environment .

background : extravehicular activity ( eva ) , such as exercise performed under unique environmental conditions , is essential for supporting daily living in weightlessness and for further space exploration like long mars mission.aim:the study was planned stress , workload , and physiological demands of simulated mars exploration.materials and methods : in this study , the six - person crew lived ( 24 hours ) for 14 days during a short - term stay at the mars desert research station . the heart rates , salivary cortisol , workload , peak oxygen uptake or maximal aerobic capacity of the crew are measured before , during and after an eva.results:data for heart rate showed the same trend as peak oxygen uptake or maximal aerobic capacity , with a maximal increase to 85% of peak . the rating of subscale showed a significant increase in eva as compared to run . salivary cortisol levels and heart rates were increased in both groups , although significant increased of cortisol levels and heart rates more in eva as compared to hill running crew members.conclusion:further study is required on large scale taken into account of limitations of this study and including other physiological and psychological parameters in mars analog environment .

Introduction Materials and Methods Results Discussion

PMC3469256

microsporidia are a group of obligate intracellular eukaryotic parasites first identified as the cause of pbrine disease of silkworms in 1857 . over the past two decades several genera and species of microsporidia were found in humans , and diagnosis and clinical management of microsporidiosis cases have improved significantly . despite these scientific advances , the epidemiology of human microsporidiosis is still unclear . most of what is now known about human microsporidiosis can be attributed to experience with patients infected with human immunodeficiency virus ( hiv ) . since 1985 and the first recognition of enterocytozoon bieneusi as an acquired immunodeficiency syndrome ( aids)-associated opportunistic pathogen , several hundred patients with chronic diarrhea attributed to this organism have been reported . cases of intestinal microsporidiosis due to e. bieneusi have been increasingly reported in other immunocompromised individuals such as organ transplant recipients , as well as in travelers , children , and elderly [ 312 ] . some reports in humans suggested that this species can produce asymptomatic infections in immunocompromised and immunocompetent individuals [ 1620 ] . in addition , e. bieneusi has been commonly identified in animals , especially mammals [ 2132 ] , and water , raising public health concerns about zoonotic and waterborne transmission of microsporidia [ 3335 ] . enterocytozoon bieneusi infections in humans are mainly detected by light microscopy of stained fecal smears , electron microscopy , and pcr - based methods . therefore , infection rates are difficult to compare because of the considerable differences in the diagnostics methods employed , the specimens analyzed , the geographical locations , the patient groups , and patients characteristics ( sex , age , socioeconomic conditions , immune status , and clinical features ) . in developed countries in north america , europe , and australia , studies involving hiv - seropositive persons with diarrhea reported rates between 2% and 78% [ 17 , 3645 ] . lower infection rates ( between 1.4% and 4.3% ) were reported in hiv - seropositive persons without diarrhea [ 17 , 39 , 43 , 45 ] . in one study of hiv - seronegative patients with diarrhea or pneumonia , spores of e. bieneusi were detected in 2.5% of urine , 11.5% of fecal , and 16.2% of sputum samples examined . in majority of the studies performed in developed countries e. bieneusi was the species detected most often , followed by e. intestinalis ; nevertheless , these findings are not in accordance with results of a study performed recently in czech republic , involving immunocompetent humans . in this study , e. bieneusi was detected four times less frequently than e. cuniculi , and e. intestinalis was identified only sporadically . also the majority of microsporidial infections ( reaching 94% ) were reported for nondiarrheal specimens . it spans a period between 1999 and 2009 , involving 856 patients ( 561 hiv - seropositive and 295-seronegative ) and respective clinical data . the authors observed that e. bieneusi was more common in the hiv - seropositive group of patients ( 7.0%39 ) than in the hiv - seronegative group ( 5.1%15 ) . in developing countries , e. bieneusi prevalence rates were reported between 2.5% and 51% in hiv - seropositive adult patients with diarrhea [ 4760 ] , and in 4.6% of patients without diarrhea . also in developing countries , in hiv - seronegative persons with and without diarrhea , e. bieneusi was detected in 5.35% to 58.1% of the fecal samples examined [ 19 , 51 , 53 , 54 , 61 , 62 ] . in studies conducted in children , e. bieneusi prevalence ranged from 17.4% to 76.9% in hiv - seropositive children with diarrhea , and from 0.8% to 22.5% in the immunocompetent or apparently immunocompetent groups of children with or without diarrhea [ 18 , 19 , 30 , 49 , 6368 ] . the recovery of spores of e. bieneusi in 0.8% of african children considered hiv seronegative , and in 5.9% of healthy orphans and 1.9% of child - care workers in asia , indicated the presence of enteric carriage of infection in immunocompetent persons in tropical countries [ 49 , 69 ] . table 1 gives an overview of the studies conducted on the prevalence of e. bieneusi infection in humans , in various areas . in industrialized nations , some studies reported e. bieneusi infection rates between 6.1 and 10% in travelers who suffered from self - limited diarrhea and returned from tropical destinations [ 6 , 8 ] . in addition , a prevalence of 17.0% of e. bieneusi infection in elderly persons was observed , leading to the speculation that age - related diminishment of the immunity might predispose these persons to microsporidial infections . a few prospective studies conducted in developed countries indicate that the prevalence of e. bieneusi in hiv - seropositive patients is progressively decreasing , probably due to the use of highly active antiretroviral therapy ( haart ) [ 70 , 71 ] . similar observations are also made recently in some developing countries . in a study conducted in brazil , comparing enteric parasitic infections in hiv / aids patients before and after the haart , a statistically significant reduction in the prevalence of these infections was observed ( 63.9% versus 24% ) . also in a 1-year longitudinal study of e. bieneusi infection in the same orphanage in bangkok , thailand , a decreasing pattern of prevalence , similar to the one observed for the incidence , was detected . microsporidia are ubiquitous in nature , and so it should come as no surprise that they can infect humans and cause clinical disease . e. bieneusi was the first microsporidia species observed by transmission electron microscopy in 1982 in villus epithelial cells in small intestinal biopsies from aids patients with diarrhea . this species is closely linked with persistent diarrhea and wasting in adults who are hiv / aids positive ; however , the outcome of follow - up studies involving children who are also hiv / aids - positive and severely malnourished children may be entirely different and warrants further study . in the last decade , several studies reported the recovery of e. bieneusi spores from children ' stools associated to different clinical spectra ( symptomatic and asymptomatic infections in hiv - seropositive and seronegative children ) , in different settings ( table 1 ) . in immunocompetent or apparently immunocompetent children , several cases of e. bieneusi infection have been described . in a study conducted in children considered hiv seronegative , attending two primary care centers in niamey , niger , e. bieneusi spores were recovered in 8 of 990 stool samples from these children ( six males and two females , with ages ranging between 3 and 26 months ) . three of the symptomatic ones presented other intestinal coinfections with giardia intestinalis ( one ) , entamoeba histolytica ( another ) , and g. intestinalis and trichomonas intestinalis ( the third child ) and complaint of diarrhea . due to these associations , the pathogenic role of microsporidia , in these children , is difficult to ascertain . although , for the other four symptomatic children , whose stools were positive for e. bieneusi , this was the only enteropathogen identified ; two had diarrhea only , one had diarrhea and vomiting , and the last one complaint of vomiting , dehydration , and fever without diarrhea . also a case of dual infection with e. bieneusi and cryptosporidium was reported in a healthy three - year - old turkish girl ( hiv seronegative ) living in germany that accompanied her family on a vacation trip to turkey , where they resided in a rural area . two months later the child developed profuse watery , nonbloody diarrhea , and nausea with recurrent vomiting , weight loss , and serious dehydration . as it happens in the above study the pathogenic role of microsporidia is difficult to ascertain in this child , due to the concomitant presence of cryptosporidium in stools . another investigation was carried out to search for microsporidian spores in the stool specimens of 344 toddlers , essentially hiv seronegative , aged 1 to 24 months , and hospitalized at a pediatric institution in tucumn , argentina . they were classified in two groups : i , made up of 222 children suffering from severe diarrheas , and ii by 122 affected by different pathologies , except gastroenteritis . the detection of microsporidia was done by light microscopy in smears of stained stools although the determination of the species involved was not done . coproparasitological and coprobacteriological studies were also carried out and the nutritional status of each child was determined . in group i , microsporidia were found in 7.2% ( 16/222 ) of the children , 4/68 ( 5.9% ) belonged to eutrophic children , and 12/137 ( 8.8% ) to undernourished children ; 8/16 positives were found to be related with other enteropathogenics . in group ii , microsporidia were detected in 8.2% ( 10/122 ) , 4/47 ( 8.5% ) in eutrophic children , 4/54 ( 7.4% ) in undernourished children , and without data in two cases ; five out of 10 positives were related with other enteropatogenics . in this study , the occurrence of intestinal microsporidia was important and did not show significant differences between toddlers with or without diarrhea , eutrophic , or undernourished children . an unusual e. bieneusi genotype ( peru 16 ) , transmitted from guinea pigs , was found in a 2-year - old immunocompetent child complaining of diarrhea , in peru . this child suffered weight loss that started almost concurrently with the episode of diarrhea at the beginning of this infection episode . this finding suggests that this species may cause short - lived gastrointestinal manifestation in immunocompetent persons . also , in a study conducted in portugal , involving 295 hiv - seronegative patients with gastrointestinal complaints , of whom 18.6% ( 55 ) were children , it was observed that microsporidians were more common in children 18.2% ( 10/55 ) , than in adults 6.3% ( 15/240 ) , and this difference was statistically significant ( p = 0.012 ) . the age of the hiv - seronegative children infected ranged from 20 months to 8 years , with a median of 3 years . the percentage of infection by e. bieneusi was similar in both age groups , 5.5% for children against 5.0 % for the adults . in the same study , the overall percentage of hiv - seronegative patients with diarrhea ( 4.5% ; 7 ) , due to e. bieneusi , was slightly smaller than that of nondiarrheal ( 5.7% ; 8) . studies have been conducted also in immunocompromised children , especially hiv - seropositive ones . in a prospective study conducted in madrid , spain , to determine the prevalence rates of microsporidiosis and other enteroparasites in 83 hiv - seropositive children treated in three hospitals of the city , e. bieneusi the infected child was 10 years old and presented nonchronic diarrhea and a cd4 count of 298/mm . in another investigation carried out in a day care treatment center and the national hospital of niamey , niger , in hiv - seropositive patients , before the introduction of highly active antiretroviral therapy ( haart ) , one child out of a group of 24 patients , all adults , was positive for e. bieneusi , by real - time pcr . a recent study conducted in , lisbon , portugal , involved 126 hiv - seropositive children , in a total of 561 hiv - seropositive patients with gastrointestinal complaints of whom 18.6% ( 55 ) were children , it was observed that microsporidians were more common in children 19.0% ( 24 ) , than in adults 12.4% ( 54 ) . the percentage of infection by e. bieneusi was higher in children ( 10.3 % ; 13 ) than in adults ( 6.0% ; 26 ) , but this difference was not statistically significant . in the same study , the overall percentage of hiv - seropositive patients with diarrhea ( 8.6% ; 29/339 ) , due to e. bieneusi , was lower than that of nondiarrheal ( 4.5% ; 10/222 ) . also here also studies have been carrying out in institutionalized and inpatient children , independent of the hiv status . a study was undertaken in dar es salaam , tanzania , to relate the protozoan infections by cryptosporidium , microsporidia , and cyclospora with the clinical features and hiv status of children , aged 1560 months , and adults hospitalized with diarrhea . included in the study there were two groups of inpatient symptomatic children : children with chronic diarrhea ( of whom 23 of 59 were hiv - seropositive ) and children with acute diarrhea ( of whom 15 of 55 were hiv seropositive ) . microsporidia spores were identified by staining methods and the species confirmed by transmission electron microscopy . enterocytozoon was identified in specimens from 2/59 children with chronic diarrhea ( one hiv seropositive ) and in 0/55 children with acute diarrhea . another study designed in bangkok , thailand , investigated the prevalence and clinical features of intestinal microsporidiosis in hospitalized hiv - seropositive and seronegative children with diarrhea . of the 95 hiv - seropositive and 87 seronegative children , 25.3% ( 24 ) and 14.9% ( 13 ) , respectively , were diagnosed with intestinal microsporidiosis . species identification of microsporidia spores by transmission electron microscopy demonstrated e. bieneusi in five cases . malnutrition was commoner in the hiv - seropositive group ( 79.2% versus 23.1% ; p = 0.003 ) . this study indicated that intestinal microsporidiosis was an important disease in both hiv - seropositive and -seronegative thai children with diarrhea . in a cross - sectional , case - control study , involving children aged < 60 months , with undetermined hiv status , undertaken in kampala , uganda , using genetic tools , a total of 17.4% of 1,779 children with diarrhea , attending a hospital , were infected with e. bieneusi compared with 16.8% of 667 control children ( without diarrhea ) . there was no significant relationship between infection with e. bieneusi and stunting , being underweight , wasting , or acute diarrhea . the authors observed that children with a high rate of excretion of spores were more likely to have diarrhea of longer duration ( p < 0.001 ) , which they hypothesized could suggest an association with more prolonged illness . it was concluded that e. bieneusi was widespread among children 336 months of age in uganda , and that there was no clear association of e. bieneusi with poor nutrition or diarrhea . the same authors , in a following study involving 243 children , aged < 60 months with persistent diarrhea ( > 14 days ) , admitted to the same hospital in kampala , uganda , using microscopy and pcr methods , screened for the presence of e. bieneusi and cryptosporidium in the children ' stools . they found that 32.9% ( 80 ) of them were excreting e. bieneusi , and 31.3% ( 76 ) were excreting cryptosporidium . ninety - one of the 243 children were hiv seropositive , of whom 76.9% ( 70 ) had e. bieneusi , versus 6.6% ( 10 ) of the 152 hiv - seronegative ( odds ratio = 47.33 ; 95% ci = 19.88 to 115.97 ) , while 73.6% ( 67 ) had cryptosporidium , versus 5.9% ( 9 ) without hiv ( odds ratio = 44.36 ; 95% ci = 18.39 to 110.40 ) . children with counts < 25% cd4 cells were more likely to have either e. bieneusi ( odds ratio = 7.42 ; 95% ci = 3.77 to 14.69 ) or cryptosporidium ( odds ratio = 6.45 ; 95% ci = 3.28 to 12.76 ) than those with higher cd4 percentages . however , only hiv status was independently associated with either e. bieneusi or cryptosporidium . among the 243 children with persistent diarrhea , 27.8% ( 67 ) were infected with both enteric pathogens , with hiv being the only independent predictor of coinfection . finally , some 81% of hiv - seropositive children with persistent diarrhea excreted one or both organisms , compared with only 10% of children with persistent diarrhea testing negative for hiv . as in their previous study on e. bieneusi there was no significant association between infection in this subpopulation and malnutrition even after controlling for hiv status and cd4 counts / percentages . once more authors concluded that e. bieneusi as well as cryptosporidium were highly prevalent in ugandan children , especially those immunocompromised by hiv / aids . also a study was conducted among hospital patients and school children in vhembe district , limpopo province , south africa , for microsporidia detection using pcr - rflp and staining methods . in the age group between 0 and 9 years , e. bieneusi was found in 50 children ( six hiv seropositive ) attending the hospital , of whom 80% ( 40/50 ) complaint of diarrhea , and in nine school children ( apparently healthy ) in the same age group , of whom 33% ( 3/9 ) had diarrhea . in a cross - sectional study of intestinal microsporidiosis conducted , this time in bangkok , thailand , in children who lived in an orphanage , stool samples were collected from 290 orphans ( 39 hiv seropositive and 251 hiv seronegative ) . stool analysis was made under light microscopy , and confirmation of species was performed using transmission electron microscopy . taking into consideration the results obtained in this study , the same authors conducted a 1-year - longitudinal study of e. bieneusi infection in the same orphanage . a total of 540 orphans and 81 child - care workers were enrolled in the study . using microscopy and pcr methods these authors found that 13.9% ( 75 ) of the orphans had e. bieneusi spores in fecal samples . only 28.6% ( 2/7 ) of the diarrheic children were infected by e. bieneusi . in this study e. bieneusi infection was significantly prevalent in children between 1 and 3 years of age with a lower incidence in the older age group , which may reflect the development of protective immunity . nine orphans ( three hiv seropositive ) were reinfected ( 4-month intervals between two positive pcrs ) in the study . thus , the authors hypothesized that protective immunity might not be fully developed after an infection in some children since reinfection occurred . asymptomatic infections with e. bieneusi have been reported in both hiv - seropositive and -seronegative adults although in children the asymptomatic cases were described , so far , only in hiv - seronegative children . in a study performed in tanzania , in four of 20 hiv - seronegative children admitted to hospital due to acute trauma or elective surgery , with no history of diarrhea in the previous 3 months , e. bieneusi spores were identified by staining methods and the species confirmed by transmission electron microscopy . all four asymptomatic children were underweight ( p < 0.01 ) . in the cross - sectional study of parasitic infection conducted in bangkok , thailand , in orphan children , spores of e. bieneusi were detected in stool specimens , by a staining method and confirmed by transmission electron microscopy , in 5.9% ( 13/221 ) of the hiv - seronegative children who had neither current illness nor history of gastrointestinal symptoms in the previous 1 month . also in kampala , uganda , e. bieneusi was detected by molecular methods in stool samples of 112 children , with undetermined hiv status , without diarrhea . in the study conducted in limpopo province , south africa , e. bieneusi asymptomatic infections were detected in 20% ( 10/50 ) of the children aged 0 to 9 years , observed in the hospital , and in 67% ( 6/9 ) of the school children enrolled in the study , by pcr methodology . in another study , in cameroon , e. bieneusi was , once more , detected in stools samples from healthy persons without symptoms , including teenagers ( 1219 years old ) and children in the age group between 0 and 11 years . the percentage of infection increased with age ; however , children had the highest parasite loads . the pathogenicity of microsporidia is still not clearly defined , and the mechanism by which they induce diarrhea has not been determined . some authors found that even though hiv - positive patients infected by e. bieneusi had more diarrhea than those noninfected , they in fact had less inflammation than the noninfected hiv - positive individuals , demonstrated by a lactoferrin test , even though some genotypes of e. bieneusi can cause chronic diarrhea [ 48 , 54 ] . enterocytozoon bieneusi can be an important cause of persistent diarrhea , intestinal malabsorption , and wasting in hiv - positive adults . mucosal damage associated with microsporidiosis is more extensive than that related to other opportunistic intestinal infections and leads to substantial malabsorption of vitamins , micronutrients , carbohydrates , and fats [ 71 , 78 , 79 ] . although microsporidiosis is common in children < 5 years of age , particularly those who live in developing countries or who are hiv - seropositive , the effects of infection on nutritional health of these vulnerable populations were not well documented until recently [ 18 , 64 , 65 , 80 ] . in a study conducted in uganda , in children 60 months of age with persistent diarrhea e. bieneusi was associated with lower rates of weight gain . children with microsporidiosis were predicted to weigh 1.3 kg less than children without microsporidiosis at 5 years of age . diarrhea and malabsorption seem to be the most common clinical problems associated with microsporidian infections , namely , e. bieneusi , reported in the literature [ 14 , 46 , 82 ] . however in some studies there were no statistically significant associations between the presence of microsporidia , namely e. bieneusi , in faecal specimens and patients with diarrhea [ 45 , 55 , 8385 ] . several studies suggest that e. bieneusi infection can remain asymptomatic suggesting that some persons may be asymptomatic carriers of these microorganisms [ 19 , 49 , 69 , 77 ] . since the identification of e. bieneusi as a pathogen in humans , it has been shown to be a major cause of chronic diarrhea in hiv - seropositive persons . some studies have tried to identify risk factors for e. bieneusi infection in humans ( table 2 ) . immunodeficiency , especially that associated with hiv / aids , low cd4 + t cells count ( 50 cells per microliter blood ) , and younger age are all considered risk factor for intestinal microsporidiosis due to e. bieneusi [ 18 , 19 , 45 , 64 , 86 , 87 ] . in ethiopia , in a study conducted in 214 hiv - seropositive and 29 hiv - seronegative patients with diarrhea , microsporidial parasites ( e. bieneusi and e. intestinalis ) were detected only in hiv - seropositive patients ( 39 ) . among the patients studied 92.3% had diarrhea for over four weeks , and 94.9% had weight loss of more than 10% . also in a recent study performed in russia , involving 159 hiv - seropositive patients , a statistically significant association was observed between the microsporidia positive patients and weight loss of > 10% of the baseline ( 63% ; 19/30 ) . in a 10-year study , performed in portugal , involved 856 ( 675 adults and 181 children ) hiv - seropositive and -seronegative patients with gastrointestinal complaints , an immunosuppressive condition and youth ( children ) were the risk factors observed for microsporidian infection . other risk factors associated with intestinal microsporidiosis , in general , or particularly to e. bieneusi infection have been reported . a us study of hiv - seropositive patients with diarrhea showed significant association of intestinal microsporidiosis with contact with horses , having been stung by a bee , hornet , or a wasp , and having used injection drugs . the use of hot tub or spa and occupational contact with water also reached statistical significance in this study . also person - to - person transmission has been suggested in an orphanage , where a multivariate analysis showed that orphans who were 1223 months old , girls , and living in one particular house were independently associated with e. bieneusi infection . all infected children presented the same e. bieneusi genotype in stools [ 5 , 19 ] . another study in zimbabwe showed association of e. bieneusi infection in hiv - seropositive patients with living in rural areas , consumption of nonpiped water , contact with cow dung , and contact with a person with diarrhea . these authors postulated the reasons for the unclear association between injection drug use and enteric microsporidiosis stating that injection drug users were as likely or more likely to contact potential infectious sources , such as contaminated water . contact with water is an important risk factor consistently associated with e. bieneusi infection in epidemiologic studies , and e. bieneusi spores have been detected in ground , surface , ditch , and crop irrigation water sources [ 33 , 105107 , 132 ] . however , no significant seasonal variation has been detected in the prevalence of intestinal microsporidiosis in hiv - seropositive patients . in a study of hiv / aids patients performed in peru , risk factors for e. bieneusi infection differed by genotype . infection with genotype peru-1 , one of the more common genotypes found , was associated with contact with duck or chicken droppings and lack of running water , flush toilet , or garbage collection . e. bieneusi has been able to cause clinical disease in immunocompromised and immunocompetent persons with different patterns , and the risk factors associated with these pathologies are partially known as a result of improvement in genetic typing and molecular epidemiology . the combination of epidemiologic studies with genotyping techniques has contributed to a better understanding of the characteristics of microsporidia that infect humans and of its reservoirs and transmission patterns . before the introduction of the molecular tools , the characterization of this important group of parasites relied exclusively on electron microscopic observations of the morphological differences in spores and/or endogenous developmental stages and host occurrence . the recent application of pcr - based molecular methods for species - specific identification and genotype differentiation of microsporidia has increased research interests in this group of microorganisms . the enhanced diagnostic capacity obtained with the molecular methods has improved the identification of human cases of microsporidiosis due to e. bieneusi , not only in developed countries [ 6 , 7 , 17 , 20 , 30 , 38 , 45 , 62 , 66 , 81 , 87 , 92 , 108 , 112 , 115 ] , but also , and especially , in developing countries where e. bieneusi may be a public health problem due to the magnitude of the hiv pandemic and poor sanitary conditions [ 18 , 19 , 30 , 47 , 48 , 5052 , 5456 , 62 , 64 , 68 , 88 , 89 , 99 , 102 ] . enterocytozoon bieneusi is a complex species with multiple genotypes and diverse hosts range and pathogenicity . since different strains can not be discriminated morphologically , typing of this species relies on molecular methods . pcr analysis of the internal transcribed spacer ( its ) of the rrna gene , a hypervariable sequence with about 243 bp long , followed by sequencing of the pcr products and comparison of obtained its sequences with those in the databases , is the standard method for genotyping e. bieneusi isolates from humans and animals . for several years these were the only genetic markers available [ 22 , 56 , 104 , 133136 ] . a few researchers genotyped e. bieneusi based on the analysis of the its by pcr - restriction fragment length polymorphism ( rflp ) [ 92 , 109 ] . and recently , two groups of researchers reported the analysis of the genbank e. bieneusi its sequence collection , using statistical methods , with the aim to obtain information about diversity , transmission , and evolution of this species [ 135 , 136 ] . these studies have identified the presence of host - adapted e. bieneusi genotypes in a variety of domestic animals and wild mammals , as well as a large group of e. bieneusi genotypes that do not appear to have host specificity [ 24 , 25 , 135 , 136 ] . e. bieneusi genotypes in humans have been shown to differ from each other in virulence and geographic distribution [ 48 , 136 ] . other authors , taking advantage of the current genome sequence surveys of e. bieneusi , identified four polymorphic microsatellite and minisatellite markers . together with the its , they became part of a multilocus sequence typing ( mlst ) technique developed for genotyping e. bieneusi that could be useful in epidemiologic investigations of e. bieneusi transmission , especially those concerning the public health significance of parasites of animal origin . the observations , obtained with the study of the its locus , were also corroborated with the phylogenetic analysis of the polymorphic microsatellite and minisatellite markers , identified by feng and collaborators in the e. bieneusi genome . these authors observed the formation of two large groups of e. bieneusi : zoonotic genotypes and host - adapted genotypes . actually , more than 90 different genotypes of e. bieneusi , identified by its sequence , have been described in humans and animals , and some genotypes have multiple names that have been reviewed in satn and fayer , 2011 who tabulated all the existing synonyms for the first time and proposed a standardized nomenclature for e.bieneusi genotypes based on the its sequences . about 34 of these genotypes have been found only in humans [ 5 , 18 , 24 , 48 , 75 , 76 , 92 , 94 , 99 , 109 , 112 , 114 , 115 ] ( table 3 ) , and 11 genotypes have been reported both in humans and animals [ 18 , 21 , 2429 , 48 , 55 , 56 , 75 , 76 , 92 , 95 , 99 , 102 , 109 , 111114 , 116 , 118127 , 129 , 139 ] ( table 4 ) . all these observations were made in populations from different geographic regions , involving all continents . in studies conducted in france involving hiv seropositive and seronegative patients infected with e. bieneusi , liguory and collaborators found five genotypes ( types i to v ) [ 109 , 112 ] . type i , synonym of genotype b , was the most prevalent genotype ( 75% ) . genotypes b ( type i ) and c ( type ii ) were also found in other populations [ 94 , 95 , 99 , 111 , 112 , 115 ] . genotype b ( type i ) even showed predisposition toward hiv / aids patients and was the only genotype found in a study performed in hiv - seropositive patients in australia . type iv , also named genotype k / peru2/ptebiii / beb5/beb5-var / cmits1 , was rare ( only in one human isolate ) . in contrast , type iv ( genotype k ) was the only genotype found in a population in cameroon . moreover , this genotype was also found in humans in other regions [ 18 , 48 , 56 , 75 , 76 , 99 , 111 , 112 ] and in various animals [ 2527 , 122125 ] . peru , in a population of 2,672 hiv / aids patients , 11 genotypes were identified ( peru-1 to peru-11 ) . the most prevalent genotypes were peru-1 ( 39% ) , synonym of genotype a , peru-2 ( 19% ) , synonym of type iv , and peru-9 ( 10% ) , also named genotype d ; the remaining eight genotypes were found in the population at much lower frequencies ( 19% ) [ 48 , 56 , 75 ] . some of these genotypes were also found in animals , including genotype a ( peru-1 ) , type iv ( peru-2 ) , peru-4 , also named genotype ebpc / e / wl13/wl17 , to peru-6 , also named pteb1/ptebvii , peru-7 , d ( peru-9 ) , peru-10 and peru-11 , also named peru-12 [ 21 , 2428 , 32 , 55 , 75 , 95 , 114 , 116 , 117 , 121126 ] . three genotypes ( a / peru-1 , peru-7 , and peru-11 ) , found until recently only in humans , were also identified in animals [ 32 , 117 ] . both genotypes d and type iv ( reported as genotype k ) were the most prevalent among patient groups from malawi and the netherlands . genotype d that was commonly detected in hiv - seropositive patients was also found in hiv - seronegative individuals , confirming its widespread nature [ 56 , 99 , 101 , 102 , 111 , 113 ] . in a study conducted in renal transplant recipients in spain , also in peru , in a prospective pediatric - cohort study of enteric parasites , e. bieneusi infection was identified in 31 of 388 ( 8% ) children . thirty of these children had infections with genotypes peru-1 ( synonym of a ) through peru-15 and peru-17 , and one child was infected with genotype peru-16 . fecal samples of all animals in the household of child infected with peru-16 were also analyzed including guinea pigs , chickens , dogs , and cats . peru-16 was identified in seven of the eight guinea pigs studied , and all the other animals were negative for microsporidia . this genotype was genetically very different from other known e. bieneusi genotypes in humans and was placed , in an independent clade , outside the cluster of genotypes considered to have broad host specificity in a phylogenetic analysis of the its sequences . the finding of genotype peru-16 in guinea pigs from unrelated households , and the close contacts between the study child and infected guinea pigs , all strongly suggested that the child probably acquired the infection from the guinea pigs in the household . this finding indicated that even some e. bieneusi genotypes that are apparently unique in some animals have zoonotic potential . one of the genotypes , peru-14 , also named genotype wl15/wl16 , was also found in animals . genotype ug2145 , identified for the first time in uganda , was the only e. bieneusi detected in a population of 1,779 children with diarrhea . some genotypes like genotypes b and c are considered host specific because they were exclusively found in humans . however , it must be kept in mind that they differ in very few base pairs from other sequences commonly found in other hosts . for instance , genotype type iv differs from b in 3 bp , and from a in just 1 bp . in contrast , some other genotypes have a zoonotic potential ( such as genotypes d and type iv , and more recently genotype a ) because they have been seen in various animals [ 18 , 2427 , 29 , 48 , 75 , 76 , 95 , 111 , 112 , 116 , 118 , 119 , 122124 , 126 , 127 , 129 , 139 ] . breton and collaborators revealed for the first time the presence of genotypes a and b in africa . these genotypes were also reported from hiv - seropositive and hiv - seronegative populations in europe [ 92 , 94 , 109 , 111 , 112 , 121 ] , peru [ 56 , 75 ] , thailand [ 5 , 114 ] , and niger . in breton and collaborators study , genotype d was found in one hiv - seropositive patient in gabon , and it was identified for the first time in isolates from three hiv - seronegative individuals in cameroon . it is considered a genotype with a broad host and geographic range , having been found in several countries in europe [ 26 , 111 , 112 , 127 ] , usa [ 24 , 119 ] , peru [ 48 , 56 , 75 ] , malawi , thailand [ 5 , 102 ] , vietnam , and korea [ 124 , 129 ] . three new genotypes caf1 , caf2 , and caf3 that are close relatives of genotypes e , type iv , and d , respectively , were found for the first time in the hiv - seropositive patients from gabon , with 10 out of 15 isolates . another new genotype , caf4 , a highly divergent genotype reported from humans , was equally present in hiv - seropositive patients in gabon and hiv - seronegative individuals in cameroon . its high frequency ( 25% ) in both countries may indicate that this genotype is common in central africa . ten hove and collaborators found nine new genotypes : six ( s1-s6 ) were from malawi , and three ( s7-s9 ) were from the netherlands . genotype s7 showed no linkage to any of the described genotypes and seems to be more related to the genotypes isolated from cattle . the patient ( hiv infection was considered unlikely ) infected with this e. bieneusi genotype was a middle - age man presenting severe diarrhea and rectal bleeding . two other new unnamed genotypes were detected in this study , af502396 and ay371283 , actually named as ue2145 and peru-8 , respectively . in a study conducted in changchun city , china , involving 40 fecal samples from diarrheal children and 180 fecal samples from animals ( 61 from pigs , 26 from dogs and 93 from cows ) , all collected in the same area around the city , zhang and collaborators found two previously reported genotypes ( genotypes i and j ) , and 10 new genotypes ( chn1 - 10 ) . genotypes i ( also named beb2/cebe ) and j ( also named beb1/cebb , pteb x ) , originally detected in cattle , were identified in both cow and human samples in this study . both genotypes coinfected with other genotypes in all positive samples . of the novel genotypes , chn1 , which was detected in five children , nine cows , and four pigs , was the most common genotype found in this study . genotypes chn3 and chn4 were also found in both human and cow samples , and genotypes chn5 - 10 were only detected in animals . the its sequences of the 10 new genotypes were highly homologous to those of genotypes i , j , type iv , and g published earlier [ 25 , 75 , 101 , 109 , 129 ] . genotypes chn1 , chn2 , and chn3 differed from j by one to four positions , while genotype chn4 is 2 bp shorter than type iv . due to the high level of similarity of the sequences , no obvious clusters related to host preference were observed . in a study that investigated the occurrence and prevalences of the microsporidial species enterocytozoon bieneusi and encephalitozoon spp . in 382 immunocompetent humans in the czech republic the sequence analyses of e. bieneusi - positive samples revealed seven different genotypes . for the first time , after so many studies performed in humans , in different geographic areas , genotypes that were previously reported only from cattle ( beb4 and ebpa ) and pigs ( pigebits5 , ebpa , and bfrmr2 ) were identified in humans , and three new genotypes were detected ( e. bieneusi cz1 to cz3 ) . the most prevalent e. bieneusi genotypes were ebpa ( in 10 individuals ) , cz3 ( in 4 individuals ) , and pigebits5 ( in three individuals ) , followed by bfrmr2 and beb4 ( each in two individuals ) . the cz1 and cz2 genotypes were identified only in single individuals . in the same study e. cuniculi genotype ii these data suggest that isolates of this group are able to switch hosts from animals to humans and should be regarded as isolates with high zoonotic potential . the high prevalences of microsporidia infection surprisingly found in different populations of immunocompetent humans in this study raise the question of whether these findings represent true infection resulting in shedding of parasites or ingested parasites that just passed through the gastrointestinal tract without establishing an infection . however , the chance of detecting the temporary passage of spores is quite low in the number of individuals sampled analyzed in this study . several studies indicate that the distribution of genotypes of e. bieneusi can vary by geographical locations , and it has been recently proposed that predominant genotypes in different geographical sites could be related to distinct sources of transmission . in a comparative study of the molecular epidemiology of microsporidiosis among hiv - seropositive patients in two separate geographical areas , niamey , niger and hanoi , vietnam , interesting results were obtained . in hanoi , two zoonotic genotypes d and e were identified . both genotypes have been previously recovered in both humans and animals [ 21 , 24 , 26 , 56 ] . these genotypes were also predominant in a study conducted in thailand in hiv - seropositive patients . thus , there is a potential zoonotic transmission of e. bieneusi in thailand and vietnam . zoonotic transmission can be from direct exposure to animals or by contamination of surface water by discharged domestic wastewater or animals . on the contrary , the niamey data highlighted a higher frequency of genotypes known to be human specific ( genotype a ) , similar to results obtained in children from an orphanage in thailand . these results suggest that transmission of microsporidiosis due to some e. bieneusi genotypes could occur through person - to - person contact . this mode of transmission could be facilitated by chronic carriage of e. bieneusi , as described in previous studies [ 49 , 140 ] . all these findings suggest different transmission modes of e. bieneusi in diverse geographic regions and in special groups in the same region . however , further studies with larger number of e. bieneusi samples are needed to confirm these findings . despite recent advances in the understanding and diagnosis of e. bieneusi , emerging microsporidian pathogens , more research is necessary to elucidate their complex epidemiology . in fact , studies that reflect true human infecting e. bieneusi prevalence are still insufficient . a few prospective studies conducted in developed countries indicate that the prevalence of e. bieneusi in hiv - seropositive patients is progressively decreasing , probably due to the use of haart . the recovery of spores of e. bieneusi in fecal samples of children considered hiv - seronegative , and in healthy ones , indicated the presence of enteric carriage of infection in immunocompetent persons in tropical countries , but also in other regions of the globe . also , the finding of asymptomatic chronic carriers of e. bieneusi emphasizes their importance in the transmission cycle , warning for the potential risk of reactivation of latent infection in cases of immunosuppression causing life - threatening disease in these individuals . the pathogenicity of microsporidia is still not clearly defined , and the mechanism by which they induce diarrhea has not been determined . even though some genotypes of e. bieneusi can cause chronic diarrhea and wasting syndrome in aids patients once the cd4 + t - lymphocyte count drops below 100 cells / mm . the recent application of pcr - based molecular methods to e. bieneusi identification and characterization has led to more reliable results compared with prevalence rates determined by light microscopy of stained biological smears , to better study the risk factors associated with these pathologies , to study the distribution of genotypes of e. bieneusi by geographical location , to improve source tracking , and to calculate the host range and pathogenic potential of an isolate . all typing studies performed until now were based on the analysis of the its sequences , the only molecular marker existent until recently . these studies identified the presence of host - adapted e. bieneusi genotypes in several animals , and e. bieneusi genotypes with no host specificity , which are considered zoonotic . some studies suggest that transmission of microsporidiosis due to some e. bieneusi genotypes could occur through person - to - person contact . this mode of transmission could be facilitated by chronic carriage of e. bieneusi , as described in some studies . besides , several microsporidian species that infect humans have been identified in animals , especially mammals , raising public health concerns about zoonotic and waterborne transmission of microsporidia . in addition , some studies indicate that even some e. bieneusi genotypes , which are apparently unique in some animals , have zoonotic potential . other studies indicate that the distribution of genotypes of e. bieneusi can vary by geographical locations and virulence . despite of the great genetic variation of e. bieneusi observed worldwide with the analyses of the its region of the rrna gene , studies with additional independent markers for e. bieneusi are highly desirable in order to clarify the genetic structure of the parasite 's populations . the discrimination among strains of e. bieneusi is very important , not only for the clarification of the reservoirs and of the transmission modes of this pathogen but also to help to explain its variations in pathogenicity that can not be answered with the study of a single locus .

a review was conducted to examine published works that focus on the complex epidemiology of enterocytozoon bieneusi infection in humans . studies on the prevalence of these emerging microsporidian pathogens in humans , in developed and developing countries , the different clinical spectra of e. bieneusi intestinal infection in children , in different settings , and the risk factors associated with e. bieneusi infection have been reviewed . this paper also analyses the impact of the recent application of pcr - based molecular methods for species - specific identification and genotype differentiation has had in increasing the knowledge of the molecular epidemiology of e. bieneusi in humans . the advances in the epidemiology of e. bieneusi , in the last two decades , emphasize the importance of epidemiological control and prevention of e. bieneusi infections , from both the veterinary and human medical perspectives .

1. Introduction 2. Prevalence of 3. Patterns of 4. Risk Factors Associated with 5. Molecular Epidemiology of 6. Conclusion

PMC4556078

parkinson 's disease ( pd ) is a neurodegenerative disorder characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta , resulting in the subsequent loss of function of the basal ganglia circuit . the molecular pathogenesis of pd is believed to be associated with mitochondrial dysfunction , oxidative stress , and activation of the apoptotic cascade . the synthetic compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( mptp ) induces permanent parkinsonism in humans via its metabolite mpp ( 1-methyl-4-phenylpyridinium ) [ 2 , 3 ] . mpp has been shown to induce a pd - like pathology in animals and cellular models by selective and potent inhibiting of complex 1 of the mitochondrial electron transport chain [ 4 , 5 ] . mpp - induced neuronal death is mediated by impairment of the mitochondrial membrane potential and opening of the mitochondrial permeability transition pore [ 6 , 7 ] . elevation in the reactive oxygen species ( ros ) level has also been involved in mpp - induced cytotoxicity [ 810 ] . activation of the apoptotic cascade may play a role in mpp - induced cell death by altering mitochondrial membrane permeability and controlling the release of cytochrome c from mitochondria [ 11 , 12 ] . caspase-3 activation by released cytochrome c has been shown to involve mpp - induced apoptosis [ 9 , 13 , 14 ] . once activated , caspase-3 will induce nuclear dna condensation and fragmentation and , ultimately , apoptosis . a number of antioxidants , such as xanthones , have been demonstrated to have a protective effect on vulnerable neurons under oxidative stress conditions [ 1618 ] . the fruit hull of mangosteen ( garcinia mangostana linn . ) , a tropical fruit , has been demonstrated to exert an antioxidative effect . alpha - mangostin was shown to induce a protective effect in cardiac reperfusion damage by attenuation of oxidative stress . neuroprotective activities of alpha - mangostin against h2o2-induced oxidative stress have been demonstrated in ng108 - 15 neuroblastoma cells . this xanthone ameliorated iodoacetate - induced cell death in primary cultures of cerebellar granule neurons by reducing ros formation . alpha - mangostin was also shown to attenuate the neurotoxicity induced by beta - amyloid oligomers in sk - n - sh neuroblastoma cells and primary rat cerebral cortical neurons [ 22 , 23 ] . the antioxidative property of alpha - mangostin is probably mediated by its modulatory effect on the activity of glutathione peroxidase . although alpha - mangostin has been reported to possess potential neuroprotective properties , there is insufficient information on its protective effects in a pd cellular model . this study aims to investigate whether alpha - mangostin could protect sh - sy5y neuroblastoma cells from mpp - induced apoptosis and the possible underlying mechanisms . the sh - sy5y human neuroblastoma cells were cultured in a 1 : 1 mixture of dulbecco 's modified eagle medium ( dmem ) and nutrient mixture ham 's f12 medium and supplemented with 10% heat - inactivated fetal bovine serum ( fbs ) , 1 mm sodium pyruvate , 0.1 mm nonessential amino acid , 1.5 g / l sodium bicarbonate , 100 units / ml penicillin , and 100 g / ml streptomycin . all media and supplements were purchased from gibco ( gaithersburg , md , usa ) . cells were maintained at 37c in a humidified atmosphere of 5% co2 . in the experiment the number of cells to be subcultured was assessed under a phase - contrast microscope based on the exclusion of trypan blue dye . the cultured cells were maintained for 2 days to allow for adhering on the plates . louis , mo , usa ) , or a combination of -mangostin and mpp according to the experiment design . sh - sy5y cells were seeded onto a 96-well plate at a density of 8 10 cells / well in 200 l of medium and incubated at 37c under 5% co2 in a humidified incubator for 2 days . after exposure to mpp ( 1000 m ) , -mangostin ( 10 m ) , or a combination of both for 24 hours , cell viability was measured by mtt ( 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ) colorimetric assay ( sigma - aldrich , st . this method was based on the reduction of tetra ring of mtt by mitochondrial dehydrogenases with nadh in the active mitochondria , yielding a blue formazan product , which can be measured spectrophotometrically . after incubation , 20 l of mtt ( 5 mg / ml ) was added to each well and the cells were cultured for another 4 hours , then medium was removed , and 100 l of dmso ( sigma - aldrich , st . louis , mo , usa ) was added to each well to dissolve the formazan . the color reaction was measured at wavelength 570 nm with a reference at 690 nm using the versamax tunable microplate reader with softmax pro software ( molecular devices , sunnyvale , ca , usa ) . sh - sy5y cells were seeded on coverslips and fixed with 4% paraformaldehyde in 0.01 m pbs ( ph 7.4 ) for 20 minutes at room temperature and then rinsed three times with pbs . after being washed with pbs , the cells were stained with 1 mg / ml hoechst 33258 ( sigma - aldrich , st . louis , mo , usa ) in pbs for 20 minutes at room temperature and then washed again . hoechst 33258 ( bisbenzimide ) preferentially binds to triplet adenine and thymine base pairs in the minor groove outside the double helix , which allows one to observe the morphological change in the nuclei of apoptotic cells . nuclear morphology was examined under a laser scanning confocal microscopy ( olympus fv1000 , olympus , tokyo , japan ) with excitation wavelength 556 nm and emission wavelength 573 nm . sh - sy5y cells were seeded onto 6-well plates at a density of 12 10 cells / well in 2 ml of medium and then incubated at 37c under 5% co2 in a humidified incubator for 24 hours . after exposure to mpp ( 1000 m ) , -mangostin ( 10 m ) , or the combination of mpp ( 1000 m ) and -mangostin ( 10 m ) for 24 hours , cells were trypsinized and centrifuged at 2,500 rpm , 4c for 5 minutes . the pellet was washed twice with cold pbs and resuspended at a concentration 1 10 cells / ml . apoptotic cells were detected using an fitc annexin - v / dead cell apoptosis kit ( molecular probes , eugene , or , usa ) . cells were incubated with 5 l of fitc annexin - v and 1 l of the 100 g / ml propidium iodide ( pi ) at room temperature for 15 minutes . the apoptotic cells were analyzed with a facscalibur flow cytometer ( bd biosciences , san jose , ca , usa ) , measuring the fluorescence emission at 530 nm ( fl1 ) and > 575 nm ( fl3 ) . both early apoptotic ( annexin - v - positive and pi - negative ) and late apoptotic ( annexin - v - positive and pi - positive ) cells were included in cell death determinations . the production of ros was determined by measuring the intensity of fluorescence emitted by cell - permeable fluorescent dyes , dihydrorhodamine 123 ( dhr123 ) and dihydroethidium ( dhe ) , purchased from invitrogen ( eugene , or , usa ) . dhr123 is oxidized to a fluorescent rhodamine-123 by intracellular hydrogen peroxide and peroxynitrite , while dhe is oxidized by superoxide anion to ethidium . sh - sy5y cells were seeded on 6-well plates at a density of 12 10 cells / well and incubated at 37c under 5% co2 in a humidified incubator for 48 hours . after exposure to mpp ( 1000 m ) , -mangostin ( 10 m ) or the combination of mpp ( 1000 m ) and -mangostin ( 10 m ) for 48 hours , cells were washed twice with hanks buffer salt solution ( hbss ) and trypsinized by 0.25% trypsin / edta and centrifuged at 2,000 rpm for 7 minutes . the pellets were incubated with 15 m dhr123 and 10 m dhe for 20 minutes at 37c in the dark . thereafter , cells were washed with hbss before cell fixation in 1% paraformaldehyde and the mean fluorescence intensity ( mfi ) of green ( fl1 , dhr ) or red ( fl2 , dhe ) fluorescence was determined using a facscalibur flow cytometer . sh - sy5y cells were seeded onto 6-well plates at a density of 12 10 cells / well and incubated at 37c under 5% co2 in a humidified incubator for 24 h. after exposure to mpp ( 1000 m ) , -mangostin ( 10 m ) or the combination of mpp ( 1000 m ) and -mangostin ( 10 m ) for 24 hours , cells were trypsinized and centrifuged at 2,500 rpm , 4c for 5 minutes . total mrna was extracted from the pellet using paris kit according to the supplier 's instructions . the quantity and purity of rna were determined by optical density measurements at od a260/a280 ratio with 1.8 or above using nanodrop 2000 spectrophotometer ( thermo fisher scientific inc . , wilmington , de , usa ) . the integrity of rna was confirmed by running 0.5 g of rna samples on 1% agarose gel . later , the cdna was synthesized from 1 g of rna using masterscript rt - pcr system ( 5 prime , gaithersburg , md , usa ) , according to the manufacturer 's instruction and stored at 20c until assay . kapa sybr fast qpcr kit ( kapa biosystems , woburn , ma , usa ) was used for real - time pcr quantification . the 20 l real - time pcr reaction mixture contained 20 ng cdna template , 10 l of 1x kapa sybr fast qpcr master mix , 200 nm of forward and reverse primers , and pcr - grade water . the sequences of the primers for the qrt - pcr are as follows : p53 : sense , 5-ggaggttgtgaggcgctgg-3 ; antisense , 5-cacgcacctcaaagctgttc-3 ; bax : sense , 5-cccgagaggtctttttccgag-3 ; antisense , 5-ccagcccatgatggttctgat-3 ; bcl-2 : sense , 5-catgtgtgtggagagcgtcaa-3 ; antisense , 5-gccggttcaggtactcagtca-3 ; -actin : sense , 5-tgcagaggatgattgctgac-3 ; antisense , 5-gaggactccagccacaaaga-3. the reaction was performed in performed in the applied biosystems 7500 real - time pcr system ( applied biosystems , foster city , ca , usa ) with the pcr cycling conditions as follows : 3 minutes enzyme activation at 95c , 40 cycles of 3 seconds initial denaturation at 95c and annealing / extension at 58c for 32 sec . melting curve analysis was performed for verify specificity of each primer after pcr to ensure amplification specificity . the threshold cycle ( ct ) number was determined and used in the comparative ct method . sh - sy5y cells were plated into 6-well plates at a density of 12 10 cells / well and incubated overnight . after exposure to mpp ( 1000 m ) , -mangostin ( 10 m ) or the combination of mpp ( 1000 m ) and -mangostin ( 10 m ) for 24 hours under 5% co2 in a humidified incubator at 37c , cells were trypsinized by 0.25% trypsin / edta and centrifuged at 4,000 rpm , 4c for 5 minutes . the pellets were washed with cold pbs , resuspended and incubated on ice for 30 minutes . then cells were vortexed and centrifuged at 15,000 rpm for 15 minutes at 4c . the supernatant was collected and total protein concentration was determined using a bca protein assay . an equal amount of 30 g proteins from each experiment group were separated by 15% sds - polyacrylamide gel electrophoresis . the membrane was blocked with a 5% skim milk for 2 hours , washed with tbst buffer , and incubated with 1 : 1000 dilution of cleaved caspase-3 primary antibodies ( cell signaling technology , denvers , ma , usa ) or 1 : 5000 dilution of monoclonal -actin primary antibodies ( sigma - aldrich , st . after washing , the membrane was incubated with 1 : 10000 dilution of hrp - conjugated goat polyclonal anti - rabbit igg ( abcam , cambridge , uk ) as the secondary antibody for cleaved caspase-3 and 1 : 5000 hrp - conjugated goat anti - mouse igg ( invitrogen , eugene , or , usa ) as the secondary antibody for -actin for 2 hours at room temperature before being developed using ecl prime western blotting detection ( ge healthcare , buckinghamshire , uk ) . the membrane was reproved with anti -actin antibody to control for equal loading of protein . the signal intensities were determined by densitometry using image - j software ( national institutes of health , bethesda , maryland , usa ) . statistical analyses were performed with one - way anova test followed by a post hoc analysis ( tukey 's multiple comparison test ) using graphpad prism 5 software for windows ( graphpad software , inc . , san diego , ca , usa ) . all values were presented as mean standard error of the mean ( mean sem ) for each group . to investigate the influence of alpha - mangostin on neuronal cell viability , we treated sh - sy5y cells with various concentrations of alpha - mangostin for 24 hours and examined cell viability with the mtt assay . exposure of sh - sy5y cells to alpha - mangostin induced a reduction in cell viability in a concentration - dependent manner . cell viability significantly decreased when cells were treated with 10 m alpha - mangostin for 24 hours , compared with that of control ( p < 0.01 ; figures 1 and 2 ) . treatment of 20 and 40 m alpha - mangostin led to loss of cell viability by more than 50% ( p < 0.001 ; figure 1 ) . when sh - sy5y cells were exposed to 2.510 m alpha - mangostin in the presence of 1000 m mpp for 48 hours , the co - treated cells showed a significant increase in cell viability in a concentration - dependent manner , compared to those treated with 1000 m mpp alone ( figure 3 ) . based on these results , 10 m alpha - mangostin was utilized in later experiments . apoptotic cells were quantified with pi and annexin - v dual staining using flow cytometry . the annexin - v / pi population consisted primarily of normal healthy cells , while annexin - v / pi cells were considered to be in the early stage of apoptosis , and annexin - v / pi cells were those considered to be in the necrosis / late apoptosis stage ( figure 4(a ) ) . after exposure to 1000 m mpp for 24 hours , the percentage of apoptotic cells increased ( p < 0.001 ; figure 4(b ) ) . cotreatment with 10 m alpha - mangostin in the presence of 1,000 m mpp for 24 hours significantly alleviated the apoptosis when compared to those treated with mpp only ( p < 0.001 ) . treatment with 10 m alpha - mangostin decreased the number of apoptotic nuclei ( figure 5 ) . alpha - mangostin significantly decreased the percentage of apoptotic nuclei when compared to mpp treatment alone ( p < 0.001 ; figure 5(e ) ) . to evaluate whether ros play an important role in the attenuation effect of alpha - mangostin on mpp - induced apoptosis in sh - sy5y cells , cells were exposed to 1000 m mpp and 10 m alpha - mangostin plus 1000 um mpp for 6 hours , and intracellular ros production was assessed as dhe and dhr123 fluorescence using flow cytometry . the result shows that cotreatment with 10 m alpha - mangostin in the presence of 1000 m mpp significantly decreased intracellular ros levels as measured by the mean fluorescence intensity ( mfi ) of dhe ( p < 0.001 ; figure 6(a ) ) and dhr123 ( p < 0.001 ; figure 6(b ) ) , compared to mpp treatment alone . expression of mrna of bax and bcl-2 was investigated using real - time quantitative rt - pcr analysis . bax expression significantly increased in 1000 m mpp - treated cells , compared to that of control cells ( figure 7(a ) ) . cotreatment of 10 m alpha - mangostin and 1000 m mpp for 24 hours significantly decreased the bax expression , compared to mpp treatment alone ( p < 0.01 ) . treatment with 1000 m mpp significantly decreased bcl-2 expression , while cotreatment with 10 m alpha - mangostin significantly increased bcl-2 expression ( p < 0.01 ; figure 7(b ) ) . as a result , bax / bcl-2 expression was significantly increased in sh - sy5y cells treated with 1000 m mpp for 24 hours ( p < 0.001 ; figure 7(c ) ) , whereas bax / bcl-2 expression was significantly lowered in cells cocultured in 10 m alpha - mangostin and 1000 m mpp ( p < 0.001 ) . the expression of p53 mrna was significantly increased in 1000 m mpp - treated sh - sy5y cells after 24-hour exposure when compared to unexposed cells ( p < 0.001 ; figure 7(d ) ) . cotreatment of 10 m alpha - mangostin and 1000 m mpp significantly reduced p53 expression when compared to cells cultured in mpp alone ( p < 0.01 ) . to investigate the effect of alpha - mangostin on the caspase-3 protein , a critical executioner of apoptosis activation of caspase-3 requires cleavage of the protein at asp175 into the activated p17 and p19 fragments . after a 24-hour treatment of sh - sy5y cells with 1000 m mpp , a significant increase in activated caspase-3 protein when sh - sy5y cells were exposed to 10 m alpha - mangostin in the presence of 1000 m mpp , the cotreated cells showed a significant decrease in activated caspase-3 compared to the mpp treatment alone ( p < 0.001 ) . the present study has , for the first time , demonstrated that alpha - mangostin rescues apoptosis in dopaminergic sh - sy5y cells treated with mpp , a cellular model of parkinson 's disease . the results suggest that cytoprotection of alpha - mangostin against mpp - induced apoptosis may be associated with the reduction of ros production , modulating the balance of pro- and antiapoptotic genes , and suppression of caspase-3 activation . mpp is a neurotoxin used to generate animal and cellular models of parkinson 's disease . mpp has been shown to increase ros in neuroblastoma cells [ 9 , 25 , 26 ] and induce superoxide anion ( o2 ) production via inhibition of mitochondrial complex i [ 10 , 27 ] . the presence of antioxidant enzymes such as superoxide dismutase protects against mpp toxicity in neuronal cell lines and dopaminergic neurons in primary culture . previous studies showed that alpha - mangostin reduced ros formation [ 21 , 24 , 30 ] . in the present study , dhe and dhr123 dhe is used extensively to monitor superoxide production , perhaps the most specific dye that is retained well by cells [ 31 , 32 ] . our results suggest that alpha - mangostin attenuated the oxidative effect of mpp through the reduction of superoxide production . dhr123 is used for the detection of peroxide and peroxynitrite ( onoo ) . in vitro , our dhr123 oxidation assay suggests that treatment with alpha - mangostin also decreased the production of h2o2 and onoo induced by mpp . ros are elevated in cells undergoing apoptosis and antioxidants have been shown to protect neuronal cells against apoptosis induced by a variety of apoptotic agents . ros activate caspase-3 and caspase-3-like proteases in various cell types including neuronal cells , leading to nuclear condensation and dna fragmentation [ 36 , 37 ] . ros also induce apoptosis , probably by decreasing expression of bcl-2 , an antiapoptotic molecule [ 9 , 38 ] . bcl-2 and bax may control the mitochondrial permeability transition pore , which can influence the passage of cytochrome c and other apoptosis - inducing factors that trigger the activation of caspase cascade and result in apoptosis . mangosteen extract has been shown to protect sk - n - sh neuroblastoma cells against an a-induced increase in caspase-3 activity . here , we showed that alpha - mangostin decreased caspase-3 activation , decreased bax mrna expression , and increased bcl-2 mrna expression induced by mpp in dopaminergic sh - sy5y neuroblastoma cells . a previous study on cisplatin - induced apoptotic death showed that alpha - mangostin attenuates the increase in p53 expression induced by cisplatin . proapoptotic bax , directly induced by p53 , can overcome the antiapoptotic effect of bcl-2 , whereas p53 can directly inhibit bcl-2 . in the present study , treatment with 1000 m mpp significantly increased the expression of p53 . as a result , increased p53 induced bax expression and inhibited bcl-2 expression , leading to the apoptosis of sh - sy5y cells . our results suggest that alpha - mangostin might reduce the effect of mpp on bax and bcl-2 expression by attenuating p53 expression . caspase-3 has been shown to be involved in the apoptotic events occurring in the mitochondrial - dependent pathway , which are associated with nuclear condensation and dna cleavage . caspase-3 also induces phosphatidylserine externalization from the internal to external leaflets of the plasma membrane . phosphatidylserine exposure on the external leaflet of the plasma membrane can be bound with annexin - v and is widely observed during apoptosis . our dna staining and pi / annexin - v dual staining supported the role of alpha - mangostin in protection against nuclear changes and phosphatidylserine externalization induced by mpp treatment in sh - sy5y neuroblastoma cells . alpha - mangostin is an antioxidant among the most abundant bioactive xanthones found in the mangosteen pericarp , which has long been used in traditional medicine to treat diarrhea , dysentery , infected wounds , and chronic ulcers . it has a broad range of bioactivities , such as antioxidant , anti - inflammation , anticancerogenic , and antihistaminergic effects [ 39 , 4447 ] . alpha - mangostin has a cardioprotective effect against myocardial infarct and reperfusion injury in rats ; such an effect was related to low levels of lipid peroxidation , a decrease in protein carbonylation and the preservation of high glutathione content [ 19 , 48 ] . recently , alpha - mangostin has been shown to inhibit and dissociate the beta - amyloid aggregation , which could contribute to its effect of attenuating beta - amyloid oligomers - induced neurotoxicity in a cellular model of alzheimer 's disease . this study indicates that alpha - mangostin protects sh - sy5y cells against mpp - induced apoptosis . the underlying mechanism could be attributed to its antioxidative properties and thus modulating the apoptotic process . the results showed that alpha - mangostin potentially possesses neuroprotective effects in a cellular model of pd . alpha - mangostin is a small lipid - soluble molecule with the potential to pass the blood - brain barrier .

in vitro studies have shown that extracts from mangosteen ( garcinia mangostana linn . ) act as antioxidants and cytoprotective agents against oxidative damage . the protective effect of alpha - mangostin , the major xanthone found in the pericarp of the mangosteen , in cellular models of parkinson 's disease ( pd ) , has not been investigated . this study aims to investigate whether alpha - mangostin could protect sh - sy5y neuroblastoma cells from mpp+-induced apoptosis . the effects of alpha - mangostin on mpp+-induced cell death were evaluated with a cell viability assay , staining for nuclear dna morphology , flow cytometry for apoptotic cells and reactive oxygen species ( ros ) production , quantitative real - time pcr for the expression of p53 , bax , and bcl-2 , and western blot analysis for cleaved caspase-3 . concomitant treatment with alpha - mangostin attenuated the effect of mpp+ on cell viability and apoptotic cell death . alpha - mangostin reduced ros formation induced by mpp+ . bax / bcl-2 expression ratio and expression of p53 were significantly lower in cells cocultured with alpha - mangostin and mpp+ . the cotreated cells showed a significant decrease in activated caspase-3 compared with mpp+ treatment alone . our data suggest that cytoprotection of alpha - mangostin against mpp+-induced apoptosis may be associated with the reduction of ros production , modulating the balance of pro- and antiapoptotic genes , and suppression of caspase-3 activation .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

PMC4227834

in vitro transcription reactions , particularly those mediated by t7 rna polymerase , have become a cornerstone of modern rna biochemistry and biophysics . these cell - free transformations facilitate the preparation of short as well as long native rna transcripts using synthetic and plasmid - derived dna templates . while of rather general utility , t7 rna polymerase requires a specific promoter for optimal transcription and tends to also be rather sensitive to sequence composition , particularly next to its consensus promoter . numerous studies have analyzed the initiation and elongation stages in these processes and , practically , identified optimal sequences at the transcript s guanosine residues are frequently found at positions + 1 and + 2 , where altered sequences typically suffer from significantly diminished transcription efficiency . in vitro prepared transcripts therefore almost exclusively possess a gtp at their 5 end , with 15 out of 17 reported promoters initiating transcription with pppg and 13 with pppgpg . a formidable contemporary challenge , which is compounded by the constraints outlined above , is the need to modify rna transcripts , typically with fluorescent probes , for diverse biochemical and biophysical applications . this problem has been tackled in several ways , including the development of orthogonal base pairing systems , which require , however , the synthesis of modified dna templates in addition to the necessary complementary modified triphosphates . the fastidious behavior of t7 rna polymerase has also limited the modification position , with most appearing remote to the promoter , where the enzymatic process is believed to be beyond its vulnerable initiation phase . replacing the initiating gtp at position + 1 with alternatives such as gmp and guanosine , some with modifications on the monophosphate or ribose , has been explored with different levels of success . transcription initiation with gtp analogs , where the heterocyclic nucleus has been altered has not , to our knowledge , been explored . this has motivated the study reported here , where newly synthesized gtp , a highly isomorphic and emissive analog , has been investigated as a gtp surrogate in t7 rna transcription reactions . the recent completion of an emissive rna alphabet , a fluorescent ribonucleoside set comprised of highly emissive purine and pyrimidine analogs , all derived from thieno[3,4-d]pyrimidine , presents unique opportunities for the generation of modified rna constructs . despite the high structural resemblance to their native counterparts , it was unclear at the onset of this project whether or not polymerases could accommodate and effectively incorporate these modified nucleosides into oligonucleotides . as discussed above , particularly challenging is the initiation of in vitro transcription reactions with gtp analogs , which is inevitable in this context . in this contribution we critically assess the ability of t7 rna polymerase to initiate rna transcription and elongate the nascent modified transcript using gtp ( 2 ) , a highly emissive and isomorphic gtp analog , and compare the results to its performance with the native triphosphate . we demonstrate that the modified nucleotide is capable of initiating and maintaining transcription reactions , leading to the formation of fully modified and highly emissive transcripts . importantly , to assess proper folding and function of rna transcripts where all g residues have been replaced with a synthetic analog , we explore modified hammerhead ribozymes , catalytic rna assemblies , which are likely to be exceedingly sensitive to such alterations . the fully modified enzyme and substrate of the hammerhead ribozyme are efficiently transcribed , and the impact of replacing all g residues with g is evaluated . we further demonstrate that the emissive transcripts can be used to monitor the ribozyme - mediated cleavage reaction in real time . the 5-triphosphate of g was synthesized from the parent nucleoside using freshly distilled pocl3 and tributylammonium pyrophosphate ( scheme 1 ) . the triphosphate was purified by ion - exchange chromatography and reverse - phase hplc ( see experimental section ) . final treatment with chelex 100 afforded the analytically pure nucleotide [ p nmr 9.98 ( d , j = 20.1 hz , p ) , 10.64 ( d , j = 18.6 hz , p ) , 22.64 ( t , j = 18.6 hz , p ) ] . reagents and conditions : ( i ) pocl3 , ( meo)3po , 0 c ; ( ii ) tributylammonium hydrogen pyrophosphate in dmf , bu3n , 0 c . transcription reactions with the analytically pure gtp ( 2 ) and t7 rna polymerase were performed to first analyze its enzymatic incorporation into short rna oligonucleotides . a short dna promoter template duplex was used to discern the ability of gtp to initiate transcription and be incorporated during the elongation phase ( figure 1 ) . the dna template 3 is terminated with a single t at the 5 end so a lone a is directed to the 3 end of the transcript . when trace amounts of -p atp are used , only successfully transcribed full length labeled rna products would be visible , whereas short failed transcripts would be undetected after gel electrophoresis ( figure 1 ) . t7 promoter and template 3 depicting the enzymatic incorporation reaction using natural ntps and gtp or gtp resulting in transcripts 4 or 5 . the t7 promoter and template 3 were annealed , and transcribed in the presence of natural ntps or with gtp replacing gtp and a trace of -p atp . a phosphorimage revealed a full length 10-mer product ( transcript 5 ) using gtp that corresponded to the natural triphosphates transcript 4 ( figure 2a ) . the overall yield of transcript 5 , containing four g residues , compared to the natural unmodified transcript 4 was 70 3% , indicating that the average individual incorporation was 91 1% . next , a large - scale transcription reaction was run and uv shadowing was used to visualize all products . comparing the transcription reactions with gtp to reactions with gtp illustrates that the desired product and abortive transcripts appear almost identical ( compare lanes 1 and 2 , respectively , in figure 2b ) . importantly , when visualizing the gel under uv illumination ( 302 nm ) , the product and initiation phase truncated transcripts are highly fluorescent ( right figure 2b ) . following extraction , the isolated yield of the g modified 10-mer ( transcript 5 ) was 78 12% compared to the native 10-mer ( transcript 4 ) , averaging 94 2% per g incorporation . the transcripts were characterized by esi ( figure s1a ) and digested using s1 nuclease and dephosphorylated . the nucleoside mixtures were subjected to hplc - ms analysis . comparing the chromatogram obtained for the native 10-mer ( 4 , figure s3b ) to that of the modified one ( 5 , figure 3 ) ( a ) small scale transcription using trace -p atp . lane 1 : control transcription reaction in the absence of gtp or gtp . lane 2 : control reaction in the presence of all natural ntps . lane 3 : reaction in the presence of equimolar concentration of gtp and gtp . incorporation efficiencies of gtp transcripts are reported with respect to transcription in the presence of gtp . all reactions were performed in triplicate , and the errors reported are the standard deviations . ( b ) large scale transcription reaction using template 3 with all natural ntps ( lane 1 and 1 ) or atp , utp , ctp , and gtp ( lanes 2 and 2 , 78 12% isolated yield ) with uv light at 254 nm ( on tlc plate ) and 302 nm ( pl ) . hplc - ms traces of the ( a ) mixture of nucleosides used as a standard and ( b ) digestion results of transcript 5 . digestion of 12 nmol of transcript was carried out using s1 nuclease for 2 h at 37 c and followed by dephosphorylation with alkaline phosphatase for 2 h at 37 c . the ribonucleoside mixture obtained was analyzed by reverse - phase analytical hplc , using a mobile phase of 06% acetonitrile ( 0.1% formic acid ) in water ( 0.1% formic acid ) over 12 min ; flow rate 1 ml / min . to test the runoff transcription of longer constructs and assess the function of the resulting transcripts , longer dna templates ( 6 and 7 ) were used to generate hammerhead ribozymes : the natural all native substrate ( s ) , the modified substrate ( g - s ) , the natural enzyme ( e ) , and the modified enzyme ( g - e ) ( figure 4 ) . these transcription reactions were executed only on a large scale , and the rna transcripts ( s , g - s , e , and g - e ) were isolated after polyacrylamide gel electrophoresis ( figure s4 ) , characterized by esi ( figure s1b and s1c ) , and then digested ( figure s3c and s3d ) . as before , the full length products and all short failed transcripts in transcription reactions using gtp are highly emissive ( figure s4 ) . t7 promoter and templates 6 and 7 depicting the enzymatic incorporation reaction using natural ntps and gtp or gtp resulting in transcripts s , thg - s , e , or thg - e . g is underlined and bolded blue in the transcripts g - s and g - e . following purification , the natural substrate ( s ) and g - substrate ( g - s ) were dephosphorylated with alkaline phosphatase and 5 labeled with t4 polynucleotide kinase according to standard protocols . the assembled ribozymes were then tested for the anticipated strand cleavage in all different combinations ( figure 5 ) , using conditions similar to those previously published , where the ribozyme cleavage reaction was initiated by mixing equal volumes of buffered solutions containing mgcl2 of substrate with enzyme . the reactions contained excess enzyme to obtain pseudo first - order kinetic rate constants ( k2 ) . single turnover reactions at 31 c contained 0.3 m substrate ( including a trace of 5-p labeled material ) and 3 m enzyme , in 50 mm tris - hcl ph 7.0 , 200 mm nacl , and 10 mm mgcl2 . the rate constants obtained for the native hh ribozyme s and e ( figure 5a ) and the substrate - modified one g - s and e ( figure 5b ) were 0.15 0.1 min and 0.12 0.1 min , respectively ( figure 6b ) . the unmodified hh enzyme e cleaved 87% and 86% of the substrates s and g - s , respectively , at 20 min . the fully modified g - containing enzyme ( g - e ) showed no cleavage of s or g - s at 31 c ( figures 5c , 5d , and 6a ) . reactions of g - e with the natural and modified substrates at slightly elevated temperatures ( 37 c ) did show a small amount of sequence specific cleavage , estimated at about 2% after 40 min ( figure s5 ) . hammerhead ribozymes and cleavage reactions of ( a ) natural substrate and enzyme ( s and e ) , ( b ) modified substrate and natural enzyme ( g - s and e ) , ( c ) natural substrate and modified enzyme ( s and g - e ) , and ( d ) modified substrate and modified enzyme ( g - s and g - e ) . ( a ) hh ribozyme cleavage reaction results were followed by p radioactive labeling of substrate strands s and g - s . s and p1 , and g - s and g - p1 indicate substrate and product strands ( figure 5 ) . all reactions were conducted at 31 c and contained 0.3 m substrate ( including a trace of 5-p labeled material ) , 3 m enzyme , 50 mm tris ph 7.0 , 200 mm nacl , and 10 mm mgcl2 . the reactions were quenched at the given times ( t in min ) and resolved by gel electrophoresis on a denaturing 20% polyacrylamide gel with 7 m urea . ( b ) initial kinetics of s and e and g - s and e. the pseudo - first - order rate constants ( k2 ) of the cleavage reactions are determined as the slope of ln(fraction cleaved ) versus time . ( c ) ribozyme - mediated cleavage curves as determined by p data for s and e and g - s and e. fraction cleaved ( s / s0 ) was determined by dividing the amount of cleaved substrate by the sum of the full length and cleaved substrate . the cleavage of g - s by the native enzyme e was monitored with nonradiolabeled material using steady - state fluorescence spectroscopy under the same conditions as the experiments performed with the radiolabeled constructs but in a slightly larger volume ( see figure s6 for absorption and emission spectra of g - s ) . mixing the substrate with the enzyme in a fluorescence cuvette at 31 c gave final concentrations of 0.3 m substrate and 3 m enzyme in 50 mm tris - hcl ph 7.0 , 200 mm nacl , and 10 mm mgcl2 . the fluorescence intensity , monitored at 450 nm , increased during the reaction ( figure 7a and 7c ) . alternatively , the fully modified enzyme g - e mixed with the native substrate s , which showed no measurable strand cleavage at 31 c when monitored using the p labeled substrate , displayed minimal fluorescence intensity changes ( figure 7b and 7d ) . the fraction cleaved of g - s from the radioactive and fluorescence experiments was normalized and showed a similar trend ( figure s7 ) . importantly , page analysis confirms the presence of two fluorescent products g - p1 and g - p2 ( see figure s8 ) . fluorescence spectra of ( a ) g - s and e ( blue ) ( excitation 470 nm , emission 425485 nm , slit widths 8 nm ) and ( b ) s and g - e ( black ) ( excitation 470 nm , emission 425485 nm , slit widths 4 nm ) , where t = 0 min and t = 20 min spectra have thicker lines . all reactions were conducted at 31 c and contained 0.3 m substrate , 3 m enzyme , 50 mm tris ph 7.0 , 200 mm nacl , and 10 mm mgcl2 . the fluorescence intensity shown at 450 nm over 20 min of the ( c ) cleavage of g - s by e ( blue ) and ( d ) mixing of s and g - e ( black ) . as has been previously demonstrated , fluorescent nucleoside analogs can tremendously facilitate the study of nucleic acid folding , recognition , and catalysis , including the enablement of real time assays . of particular significance is the modification of oligonucleotides with isomorphic nucleoside analogs , as their high similarity to their canonical counterparts and nonperturbing nature frequently results in faithful folding and function . a case in point is our recent study of emissive mrnas , where specific g residues have been surgically replaced by g using solid - phase synthesis . these modified rna constructs were recognized by the ribosome and capable of fluorescently reporting discrete steps in translation . here we set to explore the ability of gtp to support the initiation step of in vitro transcription reactions and then elongate the nascent rna transcripts to yield full length products in which all g residues are replaced by g. we then evaluate the impact of this rather dramatic modification on the function of the hh ribozyme as a prototypical functional rna system . the commonly used t7 promoters end with c , so one could not test a single g or g incorporation in g - containing transcripts where a g residue is remote to the promoter . we therefore initially used a short oligonucleotide 3 that has been previously used by our lab to evaluate t7 rna polymerase s ability to produce short modified transcripts ( figure 1 ) . transcription reactions using template 3 in the presence of gtp , ctp , atp , and utp but no gtp ( figure 2a , lane 3 ) yielded full length products , which suggested that gtp could indeed initiate such transcription reactions . the high incorporation efficiency of the g modification at an average yield of 91 1% per incorporation demonstrated its structural and functional similarity to g. it is likely , however , that the incorporation yield at positions + 1 and + 3 is lower than those at positions + 6 and + 8 , due to their proximity to the promoter . capitalizing on these positive results , the transcription reactions were scaled up to provide large quantities of full length products for analytical characterization . indeed , ms analysis and enzymatic digestion reactions followed by hplc - ms analysis further confirmed that gtp was indeed recognized by t7 rna polymerase , and efficiently utilized during the initiation and elongation phases ( figure 3 ) . the intense fluorescence of the abortive transcripts provided another positive indication that gtp was indeed the agent responsible for initiating transcription ( figure 2b ) . dna templates ( e.g. , 6 and 7 ) , yielding medium rna transcripts , also demonstrated that gtp is efficiently incorporated during the initiation and elongation phases . much longer transcripts , which include multiple g residues at the 5-end , as well as several consecutive g residues , have also been transcribed in high yields , with an average incorporation yield of 95% per gtp ( see figure s9 ) . we submit that these observations validate g as a true isomorphic nucleoside analog of g , which is faithfully recognized by the polymerase , while retaining high selectivity for wc pairing . although , in principle , multiple alterations within an rna transcript might be functionally detrimental , the intense emission of these per - modified transcripts suggests potential utility ( see below ) . additionally , established protocols in rna biochemistry allow ligations of rna fragments into larger constructs , which suggest that short g - containing rnas can be ligated to longer native ones for certain applications . in addition to gtp being a substrate for t7 rna polymerase , we tested the fully modified oligonucleotides , specifically g - s and g - e , for their ability to function as components in a hh ribozyme , as a prototypical catalytically active rna . the substrates ( s and g - s ) were first radioactively labeled to visualize their ribozyme - mediated cleavage . incidentally , this successful labeling demonstrates that the 5-end of the transcript g - s does not hamper alkaline phosphatase - mediated dephosphorylation and t4 polynucleotide kinase - mediated phosphorylation . these observations are of significance , as they suggest that modifying rna transcript with g does not hinder enzymatic transformations by commonly used molecular biological agents . the unmodified hh enzyme e cleaves its native substrate s and the fully modified one g - s with similar rates , indicating that replacing all g residues with g in the hh substrate does not substantially interfere with ribozyme catalysis . in contrast , the fully modified hh enzyme g - e showed very little cleavage ability of either the native substrate s or the corresponding modified g - s . this suggests that the substitution of g for g interferes with either the folding or catalysis of the hh enzyme . as the modified substrate is almost fully duplexed and effectively cleaved by the native enzyme , attention is then focused on the relevant residues in the enzyme that are proposed to be involved in catalysis : g8 , g10.1 , and g12 ( figure 5c ) . in the proposed ribozyme cleavage mechanism , the n7 of g10.1 coordinates a divalent metal ion , which appears to be involved in the phosphodiester cleavage reaction . the lack of the imidazole ring and hence of n7 in g may therefore explain the severely attenuated catalytic activity of the fully modified enzyme g - e . additionally , the nucleobase of g12 is involved in the cleavage reaction where the putatively deprotonated n1 position acts as a base to abstract the proton on the o2 of c17 , which then nucleophilically attacks the adjacent 3 phosphate , eventually leading to strand cleavage . since the pka of the n1 position of g within the folded ribozyme likely differs from that of g the sugar and not the nucleobase is proposed to be involved in catalysis , making it less likely that modification at this position is directly hindering cleavage . importantly , the cleavage of the modified hh substrate g - s by the native enzyme e can be observed using steady - state fluorescence spectroscopy , demonstrating the utility of such emissive transcripts for monitoring rna related processes in real time ( figure 7a and 7c ) . a significant fraction of the large increase seen in fluorescence intensity likely originates from the cleavage of g - s by e because inactive combinations ( such as that of s and g - e ) exhibit much smaller fluorescence intensity changes ( figure 7b and 7d ) . we note , however , that changes in emission intensity observed when mixing such fluorescent rna strands represent multiple events , including the annealing , folding , and mg coordination , as well as strand cleavage and dissociation . this is supported by the small fluorescence increase seen when the s and g - e are mixed to generate an inactive hh ribozyme , which likely reflect only conformational changes and metal coordination ( figure 7b and 7d ) . notably , however , when the normalized data generated by p labeling is compared to the fluorescence - generated rates for g - s and e , similar overall trends and rates are seen ( figure s6 ) . while these deficiencies can likely be circumvented by monitoring the process using a stopped - flow kinetic apparatus ( thus separating the fast events from the relatively slow cleavage reaction ) , we emphasize that monitoring catalytic rnas using simple benchtop steady - state fluorescence spectrometers can be an effective way to probe substrate cleavage and inhibition . it greatly reduces the overall experimental time , and unlike p - monitored reactions , which are not monitored in real - time , can provide additional insight into conformational changes , hybridization , and magnesium binding events , among others . gtp is found to be accepted by t7 polymerase as a faithful gtp surrogate . this highly isomorphic nucleoside triphosphate can initiate transcription , as well as be incorporated during the elongation phase in short and longer oligonucleotides . importantly , such modified transcripts , which contain a pppg at their 5-end , are also successfully dephosphorylated with alkaline phosphatase and then phosphorylated with t4 polynucleotide kinase , illustrating that other terminus - modifying enzymes tolerate this modification as well . to investigate the impact of g substitution on rna function , we tested hh ribozyme combinations where the substrate and enzyme were per - modified with g. a hh ribozyme containing a fully modified substrate g - s hybridized to a native enzyme e undergoes efficient phosphodiester bond cleavage that can be monitored either with radioactively labeled substrate followed by page or , in real time , using gs fluorescence . in contrast , the fully modified hh enzyme g - e displayed very little cleavage ability with either s or g - s . for example , the inactivity of the fully g modified hh enzyme suggests utility of this nucleoside in probing specific mechanistic questions in rna catalysis . moreover , due to their reliable hybridization and wc pairing , highly isomorphic but potentially inactive hh constructs could provide useful tools for structural analysis . as such , the observation that gtp is accepted by t7 polymerase as a faithful gtp surrogate ( in both its initiation and elongation phases ) and that other enzymes , commonly used in molecular biology , accept such per - modified strands as viable substrates opens up numerous creative opportunities to utilize such modified nucleosides and oligonucleotides , both as structural as well as fluorescent tools . even 2-aminopurine , an extensively employed emissive a isoster , fails to perform in certain cases . we feel , however , that g can be an extremely useful probe due to its unique structural features and favorable photophysical characteristics . oligonucleotides were purified by polyacrylamide gel electrophoresis and desalted on sep - pak ( waters corporation ) . ntps and the ribonuclease inhibitor ( ribolock ) were obtained from fermentas life science . radiolabeled -p atp ( 10mci / ml , 3000 ci / mmol ) and -p atp ( 10mci / ml , 6000 ci / mmol ) were obtained from perkinelmer . chemicals for preparing buffer solutions were purchased from fisher biotech ( enzyme grade ) . autoclaved 0.1% depc treated water was used in all biochemical reactions and fluorescence titrations . small molecule mass spectra ( ms ) were recorded at the university of california , san diego , chemistry and biochemistry mass spectrometry facility , utilizing an agilent 6230 hr - esi - tof mass spectrometer . reverse - phase hplc ( vydac c18 column ) purification and analysis were carried out using an agilent 1200 series instrument . steady - state fluorescence experiments were carried out in a microfluorescence cell ( 125 l ) with a path length of 1.0 cm ( hellma gmbh & co. kg , mllheim , germany ) on a horiba jobin yvon ( fluoromax-3 ) spectrometer . mass spectra for oligonuceotides were obtained on a thermofinnigan lcq deca xp at trilink biotechnologies , inc . tris(tetrabutylammonium ) hydrogen pyrophosphate ( 0.99 g , 1.1 mmol ) in a 10 ml round - bottom flask , and g ( 60 mg , 0.20 mmol ) in a 25 ml round - bottom flask , were separately coevaporated with anhydrous pyridine and dried . trimethyl phosphate ( 2 ml ) was added to g and cooled in an ice bath to 0 c . phosphoryl chloride ( 46 l , 0.5 mmol ) was added slowly , and the reaction was stirred for 2 h at 0 c , resulting in a pinkish brown solution . the coevaporated tris(tetrabutylammonium ) hydrogen pyrophosphate was dissolved in 2 ml of anhydrous dmf and added to the g reaction mixture . then tributyl amine ( 0.26 mmol , 1.1 mmol ) was added and the reaction was kept stirring at 0 c for 40 min . to the reaction mixture was added 6 ml of 1 m triethylammonium bicarbonate buffer ( teab ) , and the mixture was stirred briefly . the mixture was then transferred to a separatory funnel and washed with 10 ml of etoac . the organic layer was then back - extracted with 5 ml of 1 m teab . the aqueous layers were combined and concentrated under reduced pressure at room temperature to afford an oily yellow residue . the residue was dissolved in 10 ml of 0.05 m ammonium bicarbonate buffer and loaded onto a deae sephadex a25 anion - exchange column kept in a cold room at 4 c . the column was eluted using a gradient mixer with 0.050.5 m of ammonium bicarbonate buffer . a fraction collector was used to collect 260 fractions that were about 8 ml ( 220 drops ) . the fractions containing the triphosphate were evaporated under reduced pressure at 10 c , and then the residue was lyophilized . to purify the triphosphate further , the residue was run on another deae sephadex a25 anion - exchange column and eluted with 0.060.6 m ammonium bicarbonate buffer . after lyophilization , the triphosphate was treated with 25 mg of chelex 100 for 15 min with occasional shaking , and then filtered . the triphosphate was further purified by hplc ( phenomenex synergi fusion - rp 80a c18 column , 4 m , 250 10 nm , 04% acetonitrile in 50 mm teab buffer , ph 6.0 , 30 min ) . h nmr ( 500 mhz , d2o ) 8.10 ( d , j = 4.7 hz , 1h ) , 5.164.97 ( m , 1h ) , 4.34 ( s , 1h ) , 4.21 ( dd , j = 11.5 , 9.5 hz , 4h ) ; p nmr ( 202 mhz , d2o ) 9.98 ( d , j = 20.1 hz , p ) , 10.64 ( d , j = 18.6 hz , p ) , 22.64 ( t , j = 18.6 hz , p ) ; hr esi - ms ( negative ion mode ) [ c11h15n3o14p3s ] calculated 537.9493 , found 537.9500 ; esi - ms ( negative ion mode ) [ c11h15n3o14p3s ] calculated 538.24 , found 537.98 and 559.96 as [ m 2h + na ] . single strand dna templates were annealed to an 18-mer t7 rna polymerase consensus promoter sequence in te buffer ( 10 mm tris - hcl , 1 mm edta , 100 mm nacl , ph 7.8 ) by heating a 1:1 mixture ( 10 m ) at 90 c for 3 min and cooling the solution slowly to room temperature . transcription reactions were performed in 40 mm tris - hcl buffer ( ph 7.9 ) containing 500 nm annealed templates , 10 mm mgcl2 , 10 mm dithiothreitol ( dtt ) , 10 mm nacl , 2 mm spermidine , 1 u/l rnase inhibitor ( ribolock ) , 1 mm gtp or 1 mm gtp , 1 mm ctp , 1 mm utp , 20 m atp , 2 ci -p atp ( 800 ci / mmol stock ) , and 2.5 u/l t7 rna polymerase ( fermentas ) in a total volume of 20 l . after 3 h at 37 c , reactions were quenched by adding 10 l of loading buffer ( 7 m urea in 1 tbe with 0.05% bromophenol blue and 0.05% xylene cyanol ) , heated to 75 c for 3 min , and 10 l was loaded onto an analytical 20% denaturing polyacrylamide gel . transcription efficiencies are reported with respect to transcription in the presence of natural nucleotides . transcription efficiencies were determined from three independent reactions , and the errors reported represent standard deviations . to preparatively isolate rna and for enzymatic digestions large scale transcription reactions using template 3 were performed in a 250 l reaction volume under similar conditions , with the following changes . the reaction contained 1 mm atp , ctp , and utp , 1 mm gtp or gtp , 15 mm mgcl2 , 500 nm template , 1500 units t7 rna polymerase , and 250 units of ribolock . after incubation for 5 or 6 h at 37 c , the precipitated magnesium pyrophosphate was removed by centrifugation . the mixture was heated at 75 c for 3 min and loaded onto a preparative 20% denaturing polyacrylamide gel . the gel was uv shadowed ; appropriate bands were excised , extracted with 0.5 m ammonium acetate , and desalted on a sep - pak column . concentrations of the rna transcript were determined using absorption spectroscopy at 260 nm using the following extension coefficients : c , 7200 ; u , 9900 ; g ; 11500 ; a , 15400 ; and g , 5517 lmol cm . to preparatively isolate rna and for enzymatic digestions large scale transcription reactions using template 6 and 7 were performed in a 250 l reaction volume under similar conditions , with the following changes . the reaction contained 2 mm ctp , utp , and atp , 2 mm gtp or gtp , 20 mm mgcl2 , 500 nm template , 6 u/l ( 1500 u ) t7 rna polymerase , and 1 u/l ( 250 u ) of ribolock . after incubation for 5 or 6 h at 37 c , the precipitated magnesium pyrophosphate was removed by centrifugation . the mixture was heated at 75 c for 3 min , and loaded onto a preparative 20% or 15% denaturing polyacrylamide gel . the gel was uv shadowed ; appropriate bands were excised , extracted with 0.5 m ammonium acetate , and desalted on a sep - pak column . concentrations of the rna transcript were determined using absorption spectroscopy at 260 nm as described above . all transcripts ( 12 nmol of 4 , 5 , s , g - s , e , g - e ) were incubated with s1 nuclease in reaction buffer ( promega ) for 2 h at 37 c . the reaction was further treated with alkaline phosphatase and dephosphorylation buffer ( promega ) for 2 h at 37 c . the ribonucleoside mixture obtained was analyzed by reverse - phase analytical hplc with an agilent column eclipse xdb - c18 ( 5 m , 4.6 150 mm ) . mobile phase : 06% acetonitrile ( 0.1% formic acid ) in water ( 0.1% formic acid ) over 12 min ; flow rate 1 ml / min . mass spectra as raw data were taken by esi mass spectrometer in negative ion mode with xcalibur software version 1.3 , and the raw esi - ms m / z data were deconvoluted by promass for xcalibur version 2.5 sr-1 . the running buffer was 10 mm tert - butylamine in 70% acetonitrile in water . all deconvoluted mass spectra are in figure s1 and an example of raw m / z data and deconvoluted spectra are shown in figure s2 . the rna transcripts s and g - s ( 12.9 pmol and 12.3 pmol , respectively ) in 3 l of 10 dephosphorylation buffer and 1 l of calf intestinal alkaline phosphatase in a total volume of 30 l were incubated at 37 c for 2 h. water ( 70 l ) was added and the reaction mixture extracted with 100 l of phenol : chloroform ( chcl3):isoamyl alcohol ( iaa ) = 25:24:1 . the water layer was extracted with chloroform ( 100 l ) . the rna in the aqueous layer was precipitated with 6 l of glycoblue , 20 l of 10 m nh4oac , and 400 l of etoh and put in dry ice bath for 1 h , followed by centrifugation at 14,000 rpm for 20 min and removal of the supernatant . the pellet was washed 4 with 50 l of cold 70% etoh . the pellet was air - dried for 30 min and then dissolved in 38 l of water . five l of 10 kinase buffer , 1 l of dithiothreitrol , 5 l of -p atp , and 1 l of t4 polynucleotide kinase , were added and the reaction was heated to 37 c for 2 h. the rna was then precipitated ( 2 l of glycoblue , 10 l of 10 m nh4oac , and 200 l of etoh ) and washed ( 1 with 25 l of cold 70% etoh ) , similar to the above procedure . the pellet was dissolved on 1 tbe 7 m urea loading buffer , and then the rna was resolved by gel electrophoresis on a denaturing 20% polyacrylamide gel . the rna was cut out and extracted with water overnight , filtered , and then concentrated using a speed vac . cleavage reactions were conducted in a total reaction volume of 34 l for the natural enzyme ( e ) and 22 l for the g - enzyme ( g - e ) with the substrate ( s ) or g - substrate ( g - s ) for radiography . for the fluorescence - based experiments , the reactions were carried out at 31 c in a buffer containing 50 mm tris - hcl ( ph 7.0 ) and nacl ( 200 mm ) . buffered solutions of the substrate ( 0.6 m with traces of 5-p labeled substrate ) and enzyme ( 6 m ) were denatured separately by heating to 90 c for 90 s and cooled to room temperature over 10 min to allow for refolding . mgcl2 ( to make a final concentration of 10 mm ) was added to both the enzyme and substrate , and both were equilibrated at 31 c for 10 min . the cleavage reaction was then initiated by manually mixing equal volumes of the modified or natural substrate ( 0.6 m ) with the enzyme or modified enzyme ( 6 m ) in a heat block at 31 c , to give final concentrations of 0.3 m of the substrate and 3 m of the enzyme and 10 mm mgcl2 . for initial data points ( time = 0 ) , following initiation of the reaction , 4 l aliquots were removed at designated time periods and quenched with 12 l of urea containing loading buffer ( 7 m urea , 1 tbe , and 0.05% bromophenol blue , and xylene cyanol ff ) . the tubes were heated to 90 c for 90 s and loaded on a 20% polyacrylamide with 7 m urea gel . corresponding bands were quantified on a personal molecular imager and analyzed with quantity one software ( biorad ) . rate constants ( k2 ) were calculated as the slope of ln(1 s / s0 ) versus time , where s / s0 is the fraction of cleaved substrate . for experiments utilizing a radioactively labeled substrate , s / s0 was determined by dividing the amount of cleaved substrate by the sum of the full length and cleaved substrates .

the fastidious behavior of t7 rna polymerase limits the incorporation of synthetic nucleosides into rna transcripts , particularly at or near the promoter . the practically exclusive use of gtp for transcription initiation further compounds this challenge , and reactions with gtp analogs , where the heterocyclic nucleus has been altered , have not , to our knowledge , been demonstrated . the enzymatic incorporation of thgtp , a newly synthesized isomorphic fluorescent nucleotide with a thieno[3,4-d]pyrimidine core , is explored . the modified nucleotide can initiate and maintain transcription reactions , leading to the formation of fully modified and highly emissive rna transcripts with thg replacing all guanosine residues . short and long modified transcripts are synthesized in comparable yields to their natural counterparts . to assess proper folding and function , transcripts were used to assemble a hammerhead ribozyme with all permutations of natural and modified enzyme and substrate strands . the thg modified substrate was effectively cleaved by the natural rna enzyme , demonstrating the isomorphic features of the nucleoside and its ability to replace g residues while retaining proper folding . in contrast , the thg modified enzyme showed little cleavage ability , suggesting the modifications likely disrupted the catalytic center , illustrating the significance of the hoogsteen face in mediating appropriate contacts . importantly , the ribozyme cleavage reaction of the emissive fluorescent transcripts could be followed in real time by fluorescence spectroscopy . beyond their utility as fluorescent probes in biophysical and discovery assays , the results reported point to the potential utility of such isomorphic nucleosides in probing specific mechanistic questions in rna catalysis and rna structural analysis .

Introduction Results Discussion Conclusions Experimental Section

PMC4462794

leukodystrophies are congenital demyelinating disorders causing damage to the myelin sheath , an insulating membrane that surrounds nerve cells in the brain . there are forty different types and adrenoleukodystrophy ( ald ) is an x - linked inherited disorder with a prevalence of 1 in 20,000 - 50,000 individuals worldwide . people with x - linked adrenoleukodystrophy ( x - ald ) accumulate high levels of saturated , very long chain fatty acids ( vlcfa ) in the brain and adrenal cortex . the loss of myelin and the progressive dysfunction of the adrenal gland are the primary characteristics of x - ald . while nearly all patients with x - ald suffer from adrenal insufficiency , also known as addison 's disease , the neurological symptoms can begin either in childhood or in adulthood . the childhood cerebral form is the most severe , with onset between ages 4 and 10 . the most common symptoms are usually behavioral changes such as abnormal withdrawal or aggression , poor memory , and poor school performance . other symptoms include visual loss , learning disabilities , seizures , poorly articulated speech , difficulty swallowing , deafness , disturbances of gait and coordination , fatigue , intermittent vomiting , increased skin pigmentation and progressive dementia . there are a couple of case reports in india on this disorder , but none relating the symptoms to attention deficit hyperactivity disorder ( adhd ) . we are presenting this interesting case of x - ald , childhood onset , initially presenting to psychiatric outpatients department with features of adhd . a 7-year - old boy was brought to the psychiatry outpatient department by his mother . no concerns noted in the school or at home until 5 years of age . when he progressed to year one in school , problems started . teachers complained that he had been inattentive , does nt sit at one place and disruptive in the class . his scholastic performance declined , and mother started having difficult time at home as well . he showed no interest in homework , could nt remember what has been taught , more importantly started to forget what he has learnt in the past 2 years . the teachers have advised consulting a psychiatrist and that he may need a special school for further education as his behavior was unmanageable . she however tried changing the schools twice before bringing him for consultation , but his behavior problems got worse . during the assessment the boy was very impatient , constantly trying to leave the interview room , running up and down the stairs , distractible , and uncooperative . the assessment was done on two occasions , and neuropsychologist was involved who administered rating scales to screen and assess the severity of behavioral problems . the boy had hyper - pigmented lesions in his face , but it was overlooked at that point . he was started on methylphenidate 5 mg initially and titrated to 10 mg . as his behavior was very disruptive , putting himself at risk of accidental harm , he was started on risperidone 0.5 mg . two weeks later he presented with fever and an episode of generalized tonic clonic seizures . he presented again the following week with another episode of generalized tonic - clonic seizures with fever and altered consciousness . brain was done which revealed symmetrical t2-hyperintense white matter changes mainly in the posterior periventricular area . pediatric neurologist and endocrinologist were involved , plasma assay of vlcfa done and a diagnosis of ald was made . probing family history revealed similar problems in his cousin who died at the age of ten . literature search has shown a couple of case reports from united states where ald was misdiagnosed as adhd . this is the first time we hear about this rare neurological condition presenting as adhd in india . it never struck in our mind that symptoms of adhd could be secondary to another neurological disorder in this boy given the typical nature and onset of the presentation . furthermore , we overlooked the hyperpigmentation in the face initially until he was seen by endocrinologist after the mri helped with the diagnosis . the presence of facial hyperpigmentation and occurrence of tonic clonic seizures during the course of the illness should alert the clinicians look for these rare but important genetic cause . we explained the course and prognosis of the condition to the parents and given appropriate psychological support . the boy was also referred to special education and occupational therapy as part of the treatment plan .

x - linked adrenoleukodystrophy ( x - ald ) is one the leukodystrophies causing a progressive decline in neurological function mainly affecting the children . the most common symptoms are changes in behavior , including social withdrawal or aggression , poor memory or poor scholastic performance . here , we present a 7-year - old boy who presented with symptoms of inattention and hyperactivity and later turned out to be a case of x - ald .

INTRODUCTION CASE REPORT COURSE OF THE DISORDER DISCUSSION

PMC4199966

about 70% of stroke patients experience altered arm function , and about 40% are left with a nonfunctional arm.1 arm weakness limits the individual s activities of daily living and impacts negatively on quality of life.2 the greatest recovery in upper limb function is achieved in the 6 months following a stroke , and this period offers the best opportunity for rehabilitation interventions . the main predictor of success in physical therapy is the amount of therapy undertaken , and guidelines for patients recommend high intensity repetitive motions.3 rehabilitative therapy has focused on practicing normal movements to encourage return of relevant function and inhibit compensatory habits that may prevent further recovery . more recently , there has been an emphasis on strengthening , practice of functional tasks , and forcing use of the weak limb to complete everyday activities using constraint - induced movement therapy.46 the national stroke guidelines endorse 45 minutes of daily physiotherapy in the rehabilitation phase of stroke recovery.1 physiotherapy , however , is both labor - intensive and expensive , and service provision varies widely . at present , only 50% of people with stroke receive the rehabilitation to meet their needs during the crucial first 6 months.1 furthermore , although therapists prescribe a home exercise regimen , only about 31% of patients complete the recommended exercise.7 rehabilitation after stroke using virtual reality therapy is of increasing interest.811 a cochrane review on the effect of virtual reality on arm function found limited evidence that it may be beneficial in improving arm function and activities of daily living when compared with the same dose of conventional therapy . the authors conclusion was that present studies were too small and too few to draw conclusions and more randomized controlled trials ( rcts ) were needed.12 nintendo wii sports ( wii ) is commercially available and offers task specific training that is repetitive and motivating13,14 and may be a useful adjunct to rehabilitation.1517 wii games include tennis , golf , boxing , baseball , and bowling activities , which are played using a remote control held in the same way that real world objects such as the handle of a racket are held and manipulated . the research into its efficacy is limited , although it is being used by stroke therapists.11 pilot studies have suggested faster task performance following use of wii , with no wii-related side effects or injuries.16,17 the trial of wii in stroke ( twist ) is a multicenter , pragmatic , parallel group , rct with blinded outcome assessment , incorporating a qualitative study and cost - effectiveness analysis ( table 1 ) . the primary outcome in the rct is arm function as measured by the action research arm test ( arat ) at 6 weeks.18 the arat assesses ability to handle objects and tests grasp , grip , pinch , and gross movement of elbow and shoulder of the affected arm.19,20 secondary outcome measures include occupational performance , stroke impact , and severity . the canadian occupational performance measure ( copm ) is used to detect change in self - perception of occupational performance over time.21 the stroke impact scale ( sis ) assesses change in impairments , disabilities , and handicaps following a stroke.22 the modified rankin scale ( mrankin ) measures disability following stroke.23 these outcome measures will be applied at baseline , 6 weeks , and 6 months . arm function will be assessed at 6 months using the arat and motor activity log-14 ( mal14 ) . the mal14 ascertains how well the participant has performed day to day tasks using their affected arm.24,25 participants will be asked to complete a diary to capture a daily record of duration of arm exercises and adverse events . the diary is brief and enables the participant or carer to record daily exercise time , adverse events , and visits from the national health service ( nhs ) , private carers , and social services staff . baseline data collection will include demographic data , type of stroke using the oxfordshire community stroke project classification,26 affected arm strength using the medical research council scale of muscle strength,27 and cognitive function using the montreal cognitive assessment.28 outcome measures will be purchased ( arat , copm ) or permission to use in study will be obtained from authors ( mal14 , eq-5d 3l , mrankin , and sis ) . the qualitative study will explore participants and carers experiences of using the wii and barriers to its implementation . the health economic analysis will estimate the incremental cost per quality - adjusted life year ( qaly ) gained from the cost perspective of health care and personal social services . a total of 240 participants will be recruited to the rct across ten sites in the uk by the local research practitioners . once participants have given written informed consent , outcome measures will be assessed at baseline , 6 weeks , and 6 months . a research therapist will either install the wii in the participant s home and instruct them on the use of the wii , or will advise the participant on tailored arm exercises to be performed during the 6-week intervention phase . all participants will be instructed to perform warm - up exercises prior to tailored arm exercises or wii exercises to minimize the risk of injury . the research therapist will visit the patient prior to the 6-week assessment to collect participant s diary and wii if applicable . the research therapist will remind participants not to disclose their treatment allocation during the subsequent assessment visits by the research practitioner . participants will be telephoned by a non - blinded member of the local research team on a weekly basis to encourage them to complete their diary and enable them to express any concerns they may have had about the study . all research staff will be trained at site initiation in all outcome measures use of the trial website , case report form ( crf ) completion and safety considerations . eligible participants with arm weakness following stroke will be randomized to one of two groups . the intervention group will be instructed to undertake up to 45 minutes of exercise , in a seated position , using the wii game daily for 6 weeks at home . the wii console and game will be provided on loan to trial participants as part of the study for the duration of the intervention period . the control group will be instructed to undertake up to 45 minutes of tailored arm exercises , in a seated position , daily for 6 weeks at home . both groups will continue to receive usual care and rehabilitation as provided by their services in the area . allocation to intervention will be determined by use of a web - based randomization service , accessed by the research therapist prior to the setup visit . allocation will be stratified by center , baseline arat score ( scores 049 and 5057 ) , and previous use of a wii. the research team will visually inspect the wii and clean with antiseptic wipes prior to allocation to the next participant in the intervention group . serious adverse events will be reported by the site s principal investigator ( pi ) to the clinical trials unit ( ctu ) and reported on a monthly basis to the trial steering committee ( tsc ) , chief investigator ( ci ) , and sponsor . minor adverse events thought not to be related to the trial will not be reported . for the primary outcome of arat score at 6 weeks , the literature suggests a standard deviation of about 15 and that a clinically important effect is six points.29 for a two - tailed t - test at the 5% level and with 80% power , completed data are required on 99 patients per group . to allow for attrition a statistical analysis plan will be prepared by the study statistician , in advance of any statistical analysis of the study data . participants choosing to withdraw from either intervention arm will be followed up where possible , and reasons for withdrawal , if elicited , will be reported according to the consort ( consolidated standards of reporting trials ) guidelines.30 baseline characteristics will be summarized using suitable descriptive statistics . the principal analysis will be analysis of covariance with terms for treatment group and stratification variables . a further analysis will replace baseline arat score group ( as stratified ) by the actual arat scores in order to explore the robustness of conclusions . in each case , least - squares mean treatment differences and their 95% confidence intervals will be reported . both analyses will be repeated for the secondary outcome measure of arat at 6 months , and other continuous outcome measures . the interactions between treatment group and the stratification variables will be examined and allowed for in the analyses as appropriate . all audio recordings will be transcribed verbatim , and the transcribed interviews will be analyzed using thematic analysis.31 the field notes and observation data obtained during the interviews will be included in the analysis process . themes will be drawn from the data using constant comparison methods ; participants accounts will be compared and contrasted to enhance interpretation . if data are unclear , findings will be presented to participants to allow further comment in order to aid interpretation . the qualitative researcher will use a computer - aided program to manage the data . for trustworthiness , the lead qualitative researcher will analyze a subset of five transcripts . any unresolved disagreements will be resolved by discussion with the ci , ensuring that data will be presented in such a way as to maintain blinding of treatment group . the economic evaluation will estimate the incremental cost per qaly gained from the perspective of health and social care utilization . the costs of the wii equipment will be the price paid annuitized over a useful life of 3 years assuming the equipment could be used by eight clients per year . the main source of information on resource use will be the participant diaries which will include prompts derived from the service receipt inventory ( sri).32 the sri will also be used retrospectively at 6 months . when an assessment of need is completed during the trial , details of agreed care package will be recorded . unit costs of health and social care services will be taken from standard published sources.33,34 health - related quality of life will be assessed by eq-5d 3l35 and valued using the york tariff.36 the economic evaluation will provide within - trial estimates of the incremental cost - effectiveness ratio . sampling variation will be analyzed using probabilistic sensitivity analysis , reported as a scatterplot on the cost - effectiveness plane . sensitivity analysis to estimate the effects of parameter uncertainty will include an exploration of the sensitivity of results to differences in the costs and effects of different provisions of rehabilitation services . data will be recorded on study - specific data - collection forms ( crfs ) . persons authorized to collect and record trial data at each site will be listed on the study site delegation log and authorized by the pi . completed crfs will be checked and signed at the research site by the research practitioner or research therapist ( as applicable ) before being sent to the ctu for double - data entry onto a password - protected database accessed via the internet . the ci will be responsible for the overall running of the study and will be in regular contact with the principle investigators at the other trial centers . each trial center will have a pi , a research administrator , a research practitioner , and a research therapist to conduct this study . the ctu will coordinate trial activities and assist with overall trial management and monitoring . a data - monitoring committee will not be convened due to the nature of the intervention and short duration of the trial . the trial - management group ( tmg ) will include the ci , ctu trial coordinator , trial statistician , and other members of the team and will meet regularly to monitor progress . the tsc will include a representative from the funder and a lay person . early on in the recruitment phase , these were 1 ) that a proportion of patients were assessed as having the highest possible arat score at baseline and 2 ) the slow recruitment rate . further to this , the research therapists asked that patients with severe shoulder subluxation be excluded , as pain precluded participants from playing the wii or doing arm exercises . in response to these concerns , the tsc recommended baseline arat stratification for randomization was changed from 30 to 50 points and the mal14 be added as a secondary outcome measure at 6 months follow - up as a second outcome measure for arm function . the tsc also recommended some changes to the eligibility criteria which were implemented by the tmg : to exclude patients with symptomatic shoulder subluxation , to broaden the inclusion criteria to include patients with non - dominant weakness , and to remove the requirement for participants to have supervision during their exercises . finally , the number of participating centers was increased from seven to ten , and the tmg applied to the funder for an extension to the recruitment period in order to maximize the chances that the trial could recruit the full sample size . all changes to the protocol were approved by the sponsor , the research ethics committee , and the funder . the primary outcome in the rct is arm function as measured by the action research arm test ( arat ) at 6 weeks.18 the arat assesses ability to handle objects and tests grasp , grip , pinch , and gross movement of elbow and shoulder of the affected arm.19,20 secondary outcome measures include occupational performance , stroke impact , and severity . the canadian occupational performance measure ( copm ) is used to detect change in self - perception of occupational performance over time.21 the stroke impact scale ( sis ) assesses change in impairments , disabilities , and handicaps following a stroke.22 the modified rankin scale ( mrankin ) measures disability following stroke.23 these outcome measures will be applied at baseline , 6 weeks , and 6 months . arm function will be assessed at 6 months using the arat and motor activity log-14 ( mal14 ) . the mal14 ascertains how well the participant has performed day to day tasks using their affected arm.24,25 participants will be asked to complete a diary to capture a daily record of duration of arm exercises and adverse events . the diary is brief and enables the participant or carer to record daily exercise time , adverse events , and visits from the national health service ( nhs ) , private carers , and social services staff . baseline data collection will include demographic data , type of stroke using the oxfordshire community stroke project classification,26 affected arm strength using the medical research council scale of muscle strength,27 and cognitive function using the montreal cognitive assessment.28 outcome measures will be purchased ( arat , copm ) or permission to use in study will be obtained from authors ( mal14 , eq-5d 3l , mrankin , and sis ) . the qualitative study will explore participants and carers experiences of using the wii and barriers to its implementation . the health economic analysis will estimate the incremental cost per quality - adjusted life year ( qaly ) gained from the cost perspective of health care and personal social services . a total of 240 participants will be recruited to the rct across ten sites in the uk by the local research practitioners . once participants have given written informed consent , outcome measures will be assessed at baseline , 6 weeks , and 6 months . a research therapist will either install the wii in the participant s home and instruct them on the use of the wii , or will advise the participant on tailored arm exercises to be performed during the 6-week intervention phase . all participants will be instructed to perform warm - up exercises prior to tailored arm exercises or wii exercises to minimize the risk of injury . written instructions for both groups the research therapist will visit the patient prior to the 6-week assessment to collect participant s diary and wii if applicable . the research therapist will remind participants not to disclose their treatment allocation during the subsequent assessment visits by the research practitioner . participants will be telephoned by a non - blinded member of the local research team on a weekly basis to encourage them to complete their diary and enable them to express any concerns they may have had about the study . all research staff will be trained at site initiation in all outcome measures use of the trial website , case report form ( crf ) completion and safety considerations . eligible participants with arm weakness following stroke will be randomized to one of two groups . the intervention group will be instructed to undertake up to 45 minutes of exercise , in a seated position , using the wii game daily for 6 weeks at home . the wii console and game will be provided on loan to trial participants as part of the study for the duration of the intervention period . the control group will be instructed to undertake up to 45 minutes of tailored arm exercises , in a seated position , daily for 6 weeks at home . both groups will continue to receive usual care and rehabilitation as provided by their services in the area . allocation to intervention will be determined by use of a web - based randomization service , accessed by the research therapist prior to the setup visit . allocation will be stratified by center , baseline arat score ( scores 049 and 5057 ) , and previous use of a wii. the research team will visually inspect the wii and clean with antiseptic wipes prior to allocation to the next participant in the intervention group . serious adverse events will be reported by the site s principal investigator ( pi ) to the clinical trials unit ( ctu ) and reported on a monthly basis to the trial steering committee ( tsc ) , chief investigator ( ci ) , and sponsor . minor adverse events thought not to be related to the trial will not be reported . for the primary outcome of arat score at 6 weeks , the literature suggests a standard deviation of about 15 and that a clinically important effect is six points.29 for a two - tailed t - test at the 5% level and with 80% power , completed data are required on 99 patients per group . to allow for attrition a statistical analysis plan will be prepared by the study statistician , in advance of any statistical analysis of the study data . participants choosing to withdraw from either intervention arm will be followed up where possible , and reasons for withdrawal , if elicited , will be reported according to the consort ( consolidated standards of reporting trials ) guidelines.30 baseline characteristics will be summarized using suitable descriptive statistics the principal analysis will be analysis of covariance with terms for treatment group and stratification variables . a further analysis will replace baseline arat score group ( as stratified ) by the actual arat scores in order to explore the robustness of conclusions . in each case , least - squares mean treatment differences and their 95% confidence intervals will be reported . both analyses will be repeated for the secondary outcome measure of arat at 6 months , and other continuous outcome measures . the interactions between treatment group and the stratification variables will be examined and allowed for in the analyses as appropriate . all audio recordings will be transcribed verbatim , and the transcribed interviews will be analyzed using thematic analysis.31 the field notes and observation data obtained during the interviews will be included in the analysis process . themes will be drawn from the data using constant comparison methods ; participants accounts will be compared and contrasted to enhance interpretation . if data are unclear , findings will be presented to participants to allow further comment in order to aid interpretation . the qualitative researcher will use a computer - aided program to manage the data . for trustworthiness , the lead qualitative researcher will analyze a subset of five transcripts . any unresolved disagreements will be resolved by discussion with the ci , ensuring that data will be presented in such a way as to maintain blinding of treatment group . the economic evaluation will estimate the incremental cost per qaly gained from the perspective of health and social care utilization . the costs of the wii equipment will be the price paid annuitized over a useful life of 3 years assuming the equipment could be used by eight clients per year . the main source of information on resource use will be the participant diaries which will include prompts derived from the service receipt inventory ( sri).32 the sri will also be used retrospectively at 6 months . when an assessment of need is completed during the trial , details of agreed care package will be recorded . unit costs of health and social care services will be taken from standard published sources.33,34 health - related quality of life will be assessed by eq-5d 3l35 and valued using the york tariff.36 the economic evaluation will provide within - trial estimates of the incremental cost - effectiveness ratio . if appropriate , modelling beyond the term of the trial will be undertaken . sampling variation will be analyzed using probabilistic sensitivity analysis , reported as a scatterplot on the cost - effectiveness plane . sensitivity analysis to estimate the effects of parameter uncertainty will include an exploration of the sensitivity of results to differences in the costs and effects of different provisions of rehabilitation services . data will be recorded on study - specific data - collection forms ( crfs ) . persons authorized to collect and record trial data at each site will be listed on the study site delegation log and authorized by the pi . completed crfs will be checked and signed at the research site by the research practitioner or research therapist ( as applicable ) before being sent to the ctu for double - data entry onto a password - protected database accessed via the internet . the ci will be responsible for the overall running of the study and will be in regular contact with the principle investigators at the other trial centers . each trial center will have a pi , a research administrator , a research practitioner , and a research therapist to conduct this study . the ctu will coordinate trial activities and assist with overall trial management and monitoring . a data - monitoring committee will not be convened due to the nature of the intervention and short duration of the trial . the trial - management group ( tmg ) will include the ci , ctu trial coordinator , trial statistician , and other members of the team and will meet regularly to monitor progress . early on in the recruitment phase , the tmg reported two emerging issues of concern to the tsc . these were 1 ) that a proportion of patients were assessed as having the highest possible arat score at baseline and 2 ) the slow recruitment rate . further to this , the research therapists asked that patients with severe shoulder subluxation be excluded , as pain precluded participants from playing the wii or doing arm exercises . in response to these concerns , the tsc recommended baseline arat stratification for randomization was changed from 30 to 50 points and the mal14 be added as a secondary outcome measure at 6 months follow - up as a second outcome measure for arm function . the tsc also recommended some changes to the eligibility criteria which were implemented by the tmg : to exclude patients with symptomatic shoulder subluxation , to broaden the inclusion criteria to include patients with non - dominant weakness , and to remove the requirement for participants to have supervision during their exercises . finally , the number of participating centers was increased from seven to ten , and the tmg applied to the funder for an extension to the recruitment period in order to maximize the chances that the trial could recruit the full sample size . all changes to the protocol were approved by the sponsor , the research ethics committee , and the funder . twist is the first uk rct assessing the feasibility , cost effectiveness , and acceptability of wii in stroke rehabilitation .

introductionmany stroke patients experience loss of arm function requiring rehabilitation , which is expensive , repetitive , and does not always translate into real life . nintendo wii sports ( wii ) may offer task - specific training that is repetitive and motivating . the trial of wii in stroke ( twist ) is designed to investigate feasibility , efficacy , and acceptability using wii to improve affected arm function for patients after stroke.methodthis is a randomized controlled trial ( rct ) , incorporating a qualitative study and health economics analysis that compares playing wii versus arm exercises in patients receiving standard rehabilitation in a home setting within 6 months of stroke with a motor deficit of less than 5 on the mrc ( medical research council ) scale ( arm ) . in this study , we expect to randomize 240 participants.outcome measuresprimary outcome is change in affected arm function at 6 weeks follow - up in intervention and control group using the action research arm test . secondary outcomes include occupational performance using the canadian occupational performance measure , quality of life using the stroke impact scale , cost effectiveness analysis , and a qualitative study investigating factors that influence use of wii for patients and carers.conclusiontwist is the first uk rct assessing the feasibility , cost effectiveness , and acceptability of wii in stroke rehabilitation . the trial has been registered with isrctn 06807619 and uk crn 11030 . results of the study will be published after completion of study in august 2014 .

Introduction Methods Outcome measures Recruitment and flow through study Trial intervention Randomization Equipment check Adverse-event recording Sample size Statistical analysis Analysis of nested qualitative study Cost-effectiveness evaluation Data collection Study oversight Protocol amendments Conclusion

PMC4739461

a common and serious complication of type 1 diabetes mellitus ( t1 dm ) is diabetic autonomic neuropathy [ 1 , 2 ] . cardiovascular autonomic neuropathy ( can ) is a subset of diabetic autonomic neuropathy characterized by impaired autonomic control of the cardiovascular ( cv ) system . for instance , can has been reported to increase the mortality of diabetic patients by a factor of 3.45 . clinically , the most common methods for assessing can are heart rate variability ( hrv ) analysis and baroreflex sensitivity ( brs ) [ 3 , 5 , 6 ] . in t1 dm , aspects of the baroreflex arc can be impaired , such that both baroreceptor activity and excitability are blunted [ 8 , 9 ] and the aortic depressor nerves undergo axonal atrophy . as well , autonomic efferents , primarily of the parasympathetic nervous system ( psns ) , have decreased activity , reduced responsiveness , and decreased neurochemical activity in the heart [ 10 , 11 ] . impairment of central nervous system regions has also been reported as the limiting factor of brs [ 12 , 13 ] . reduced heart rate variability ( hrv ) is often the earliest symptom of can . whether measured by time domain analysis or by frequency domain analysis and whether in clinical or experimental t1 dm , hrv is consistently reported to be reduced in t1 dm [ 5 , 1417 ] . exercise has been demonstrated to be an effective means of improving deficits in hrv and brs in both clinical and experimental t1 dm [ 1821 ] . such improvements have been attributed to improved insulin sensitivity , increased endogenous antioxidant and anti - inflammatory mediators , and improved autonomic control of the cv system [ 2224 ] . despite similar reductions in hrv and brs , there are marked differences in early - stage changes to other cv parameters between clinical and experimental t1 dm . specifically , in clinical t1 dm , increases in heart rate ( hr ) and blood pressure ( bp ) are commonly reported in early autonomic neuropathy [ 1 , 3 , 14 , 24 , 2628 ] . in contrast , experimental stz - induced t1 dm is regularly associated with decreased bp and hr , beginning shortly after diabetes induction [ 15 , 16 , 2931 ] . due to these opposing initial changes in bp and hr , exercise training is often observed to produce contrasting outcomes on cv parameters in experimental and clinical t1 dm , namely , increased bp and hr in experimental t1 dm and decreased bp and hr in clinical t1 dm [ 19 , 25 , 3234 ] . as a result , both the increase and decrease of these cv factors are concurrently cited as exercise - mediated improvements to can with little consideration of the fact that the changes are opposed between these two contexts of t1 dm . this is important because if animal models do not accurately reproduce t1 dm pathology , then the outcomes of experimental studies may not translate to the treatment of human can , as the mechanisms underlying the pathology and exercise modifications may differ . another important difference between experimental and clinical t1 dm is the common omission of insulin treatment in experimental diabetes leading to severe hyperglycemia ranging from roughly 17 to 25 mm blood glucose concentrations ( [ bg ] ) [ 15 , 16 , 29 , 30 ] . as the severity and duration of hyperglycemia have been shown to influence the degree of diabetic neuropathy , acute and steep elevations of [ bg ] in stz - induced t1 dm may not only cause early onset neuropathy to the psns but also cause acute neuropathy of the sympathetic nervous system ( sns ) and directly affect the sinoatrial ( sa ) node . these changes may mediate the observed reduction in bp and hr that arise acutely in experimental t1 dm and in late - stage clinical t1 dm [ 2 , 3538 ] . indeed , intensive insulin therapy has been shown to restore bp and hr to non - t1 dm levels in stz - induced t1 dm rats [ 25 , 39 ] . yet , despite the use of insulin therapy in clinical t1 dm , it is often the case that chronic , moderate hyperglycemia is maintained as a result of difficulties in regulating [ bg ] in response to dynamic influences on glycemic control , such as food intake and exercise [ 40 , 41 ] . this is often resultant of a tendency to err on the side of moderate hyperglycemia in order to circumvent the acute discomfort and danger associated with hypoglycemic episodes , which occur more frequently with diabetic neuropathy due to the impairment of the glucagon response [ 40 , 42 , 43 ] . to address this , our laboratory established a model of t1 dm using a multiple low - dose stz - treatment and insulin therapy to replicate the moderate hyperglycemia observed in clinical t1 dm . in our previous studies that employed this model , we observed impairments in glucose tolerance , vascular responsiveness , cardiac function , and bone health , which were improved with high intensity aerobic exercise training [ 4447 ] . the purpose of the current study was to investigate whether our model of multiple low - dose stz - induced t1 dm with insulin therapy would induce deficits in cardiovascular autonomic function more representative of clinical t1 dm , and if high intensity aerobic training could prevent those deficits . we hypothesised that ( 1 ) our model of stz - induced t1 dm would elicit indices of can , including a blunted brs ( bradycardia and tachycardia response ) , lowered hrv and intrinsic heart rate , increased vascular sympathetic tone , and increased mean arterial pressure , and ( 2 ) high intensity aerobic exercise training would prevent or ameliorate the indications of can . the protocols used in this investigation were approved by the university of western ontario council on animal care and conformed to the guidelines of the canadian council on animal care . eight - week - old male sprague - dawley rats were obtained from charles river laboratories canada ( saint - constant , quebec ) . the rats were housed in pairs and maintained on a 12-hour dark / light cycle at a constant temperature ( 20 1c ) and relative humidity ( 50% ) . sixty - four rats were randomly assigned to one of four groups as follows : ( 1 ) sedentary control ( c , n = 16 ) ; ( 2 ) exercised control ( cx , n = 16 ) ; ( 3 ) sedentary t1 dm ( d , n = 16 ) ; ( 4 ) exercised t1 dm ( dx , n = 16 ) . all functional and blood endpoint measures were acquired 24 hours after the final exercise bout . upon arrival rats subsequently , t1 dm was induced over five consecutive days by multiple intraperitoneal ( ip ) injections of 20 mg / kg streptozotocin ( stz , sigma - aldrich ) dissolved in a citrate buffer ( 0.1 m , ph 4.5 ) . diabetes was confirmed by blood glucose measurements greater than or equal to 18 mm on two consecutive days . if necessary , subsequent 20 mg / kg stz injections were administered until diabetes was confirmed . following the confirmation of diabetes , insulin pellets ( 1 pellet ; 2 u insulin / day ; linplant , linshin canada , inc . , insulin pellet doses were then monitored for 1 week and adjusted ( 0.5 pellets ) in order to obtain daily nonfasting blood glucose concentrations in the moderate hyperglycemic range of 917 mm . insulin dose was determined by multiplying the total quantity of pellet implanted ( 0.5 pellet increments ) by the amount of insulin released per pellet ( 2 units of insulin / day / pellet ) divided by the body weight ( kg ) of the rat . blood was obtained from the saphenous vein by venous puncture with a 30-gauge needle and measured via freestyle lite blood glucose monitoring system ( abbott diabetes care inc . , intravenous glucose tolerance tests ( ivgtt ) were performed on all animals prior to t1 dm induction ( pre - t1 dm ) and at the end of week 10 of the exercise training period . rats were fasted for approximately 8 to 12 hours prior to the assay and did not perform exercise on the day of their ivgtt . a sterile - filtered dextrose solution ( 50% dextrose , 50% ddh2o ) was injected ( 1 g / kg ) into the lateral tail vein of the conscious rat . following dextrose infusion , blood glucose was measured at 5 minutes , at 10 minutes , and then at 10-minute intervals thereafter until blood glucose levels plateaued . prior to the initiation of the exercise training program , rats were familiarized with the exercise equipment on two consecutive days . the familiarization consisted of two 15-minute sessions of running at progressive treadmill speeds up to 30 meters per minute ( m / min ) . the treadmill was a custom - built apparatus fabricated by the physical plant at university of western ontario and has been used in many previous studies [ 4447 ] . the exercise training program consisted of 1 hour of motor - driven treadmill running per day at 27 m / min with a 6-degree incline , 5 days per week , for 10 weeks . the exercise intensity was determined based on earlier research that investigated oxygen uptake in rats at various treadmill speeds . the chosen intensity was found to represent approximately 7585% vo2max [ 48 , 49 ] . to achieve a surgical plane of anesthesia , rats were placed in an induction chamber circulating 4% isoflurane ( 96% o2 ) . once motor reflexes were undetectable , rats were transferred to a nosecone delivering 3% isoflurane ( 97% o2 ) and placed on a hot water pad ( 37c ) . rats were cannulated with saline - infused polyethylene ( pe90 ) catheters in the right jugular vein and carotid artery and each catheter was attached to a three - way stopcock . the jugular vein catheter was used for drug infusions and the carotid artery catheter was connected in series with a pressure transducer ( px272 , edwards life sciences , irvine , california , usa ) for arterial blood pressure measurements . at the end of the preparative surgery , rats were injected ip with a 25 mg / kg cocktail of urethane ( 16 mg / ml ) and -chloralose ( 100 mg / ml ) , an anesthetic cocktail that has been shown to have the least inhibition of baseline cv control and autonomic function . a total of 10 ml of urethane/-chloralose was made , 5 ml of which was diluted to 50% with ddh2o and was used as needed to maintain anesthesia throughout data collection . isoflurane anesthesia was gradually removed , whereby urethane/-chloralose was the primary anesthesia used during data collection . heart rate ( hr ) , systolic blood pressure ( sbp ) , and mean arterial pressure ( map ) were determined from the blood pressure pulse waveform and were collected while the rats were under urethane/-chloralose anesthesia in the supine position . data were obtained using a powerlab data acquisition system , digitized , and recorded at 1000 hz using the bundled labchart 7 pro software ( adinstruments , colorado springs , co , usa ) . prior to drug infusions , 5 minutes of spontaneous electrocardiogram data was sampled at 1000 hz and analyzed with labchart hrv analysis software ( adinstruments ) . time domain analysis of the standard deviation between normal peak pulses of the pressure pulse waveform ( sdnn ) was quantified as a measure of the total variability of the hr . frequency domain analysis of the high frequency ( hf ) band of the fast fourier transform ( fft ) of the data was assessed as an index of parasympathetically mediated hrv . baroreflex sensitivity ( brs ) was assessed using the modified oxford technique [ 51 , 52 ] . the brs was quantified using the slope of the linear regression line representing the linear portion of the sigmoidal heart rate - systolic blood pressure relationship ( hr { bpm}/sbp { mmhg } ) after rapid bolus injections ( ~5 s ) of phenylephrine ( pe , 12 g / kg , 10 g / ml ) and sodium nitroprusside ( snp , 60 g / kg , 110 g / ml ) dissolved in ddh2o . ( 2007 ) . for each drug , the catheter was first filled with a 0.2 ml volume to ensure accuracy of the drug dose . after a stable baseline was obtained , a bolus injection of snp was rapidly infused and the reflex sns mediated tachycardia response was measured . the analysis began at the onset of sbp decrease after snp infusion and ended when sbp reached its nadir . this was followed by a saline flush to washout any remaining snp in the catheter . after a stable baseline was reestablished , this same procedure was then followed using pe to measure psns mediated reflex bradycardia , except that analysis began at the onset of sbp increase and ended when sbp reached its zenith . responses to pe and snp were plotted separately and only regression lines ( slopes ) with correlation coefficients ( r ) 0.70 and p < 0.05 were accepted [ 53 , 54 ] . to measure the sympathetic contribution to baseline vascular resistance , map was assessed before and after a bolus injection of the -adrenergic receptor blocker , prazosin ( 85 g / kg , 500 g / ml ) . following this protocol to ensure physiological testing did not confound serum neuropeptide y concentration [ npy ] measurement , a subset of animals from each group did not undergo surgery for heart rate variability , baroreflex sensitivity , mean arterial pressure , or vascular sympathetic tone measurements . rather , at the end of the 10-week exercise training period these animals were anesthetized via intraperitoneal injection of sodium pentobarbital ( 65 mg / kg ) and blood serum samples for [ npy ] measurement were collected upon euthanasia . serum [ npy ] was measured using an npy elisa kit ( uscn life sciences inc . ) according to the manufacturer 's instructions . following the euthanasia of animals for blood [ npy ] measurement , hearts were extracted and immediately arrested by placing them in ice cold krebs - henseleit buffer ( khb ) . hearts were cannulated for unpaced retrograde aortic constant flow perfusion ( 15 ml / min ) of coronary arteries with khb ( containing 120 mm nacl , 4.63 m kcl , 1.17 mm kh2po4 , 1.25 mm cacl2 , 1.2 mm mgcl2 , 20 mm nahco3 , and 8 mm glucose gassed with 95% o2 and 5% co2 ) that was maintained at 37c . body weight and blood glucose concentrations were compared using a two - way repeated measures anova , while endpoint measures were compared by two - way anova , with the exception of endpoint insulin dose , which was compared using a one - tailed t - test . when significance was found , pairwise comparisons were made using the fisher lsd post hoc test . data are represented as mean standard error , with a significance level set at p < 0.05 . all groups increased in body weight over the course of the study ( p < 0.05 , figure 1(a ) ) . at the end of the study , the body weights of the t1 dm groups ( d and dx ) were lower than non - t1 dm groups ( c and cx ) , and exercised groups ( cx and dx ) weighed less than their nonexercised counterparts ( c and d ; p < 0.05 ) . following the confirmation of diabetes , weekly [ bg ] was mostly maintained in the targeted range of 917 mm ; however , the [ bg ] did move outside of this range periodically . the [ bg ] in the t1 dm groups were elevated in comparison to the non - t1 dm groups ( p < 0.05 ; figure 1(b ) ) . within the non - t1 dm and t1 dm groups , there was no difference in [ bg ] between nonexercised and exercised groups ( c versus cx and d versus dx ; p > 0.05 ) . the glucose clearance rate ( kg ) of the diabetic groups ( d and dx ) decreased from pre - t1 dm to week 10 of training ( p < 0.05 ) , whereas kg of the cx group increased ( p < 0.05 ; figure 2(a ) ) . both diabetic groups had significantly lower kg values than both the control groups ( c and cx ) at week 10 ( p < 0.05 ; figure 2(a ) ) . the amount of insulin supplementation that the dx group received was significantly less than the amount the d group received at week 10 ( p < 0.05 ; figure 2(b ) ) . for resting hr and map , there was not a significant difference between groups at week 10 ( figures 3(a ) and 3(b ) , resp . ) . however , for the intrinsic heart rate ( ihr ) , there was main effect of both exercise and t1 dm , where t1 dm decreased the ihr , while exercise increased ihr ( p < 0.05 , figure 3(c ) ) . further , within the t1 dm groups ( d and dx ) , exercise increased ihr , while within the nonexercised groups ( c and d ) t1 dm decreased ihr ( p < 0.05 ) . total hrv at week 10 , as measured by the standard deviation of the normal pulse wave peaks ( sdnn ) , was not significantly different between groups ( figure 4(a ) ) . however , there was a main effect of exercise on the hf contribution to hrv , where exercise increased hf hrv ( p < 0.05 , figure 4(b ) ) . particularly , within the t1 dm groups ( d and dx ) , exercise increased hf hrv ( p < 0.05 ) . in response to snp infusion , there was not a significant difference between groups in the tachycardia brs response ( figure 5(a ) ) . however , a significant interaction between t1 dm and exercise was observed for brs during the bradycardia response to phenylephrine ( p < 0.05 , figure 5(b ) ) . more specifically , within the t1 dm groups ( d and dx ) exercise prevented the reduction in brs that was observed in the d group ( p < 0.05 ) . an interaction between t1 dm and exercise was observed for the prazosin - induced change in map ( p < 0.05 , figure 6(a ) ) . within the nonexercised groups ( c and d ) , t1 dm resulted in an increased change in map ( p < 0.05 ) . within the t1 dm groups ( d and dx ) exercise prevented the increased change in map observed in the d group ( p < 0.05 ) . there was also a main effect of t1 dm on [ npy ] ( p < 0.05 , figure 6(b ) ) . within the nonexercised groups ( c and d ) and exercised groups ( cx and dx ) , serum [ npy ] this study demonstrated that a multiple low - dose stz model with moderate hyperglycemia , maintained using insulin therapy , produced deficits in cardiovascular autonomic function without inducing the resting bradycardia or hypotension typical of other stz models . this study also showed that high intensity aerobic exercise training can prevent deficits of cardiovascular autonomic function caused by t1 dm . furthermore , because [ bg ] was held within a moderate hyperglycemic range , the observed exercise - mediated improvements to indications of can were independent of changes in [ bg ] and , instead , may primarily have been the result of improvements to other aspects of glucoregulation and/or the preservation of autonomic nervous system function . although we found time domain analysis of total hrv , as measured by the sdnn , did not demonstrate differences between groups , frequency domain analysis exposed a reduction in the hf power in the d group compared with the dx group . since the hf power corresponds to the level of vagally mediated parasympathetic hrv , these results demonstrate not only the detrimental effects of t1 dm on autonomic cardiac control but also the benefits of exercise training toward ameliorating those effects . these findings are similar to those of other experiments of both experimental [ 16 , 21 ] and clinical diabetes [ 18 , 56 , 57 ] . ( 2009 ) reported that stz - induced t1 dm reduced the hf component of hrv , which was improved by exercise . also , they found that the vagal tonus of the control exercised rats did not differ from sedentary controls . likewise , chen et al . ( 2008 ) reported that children with t1 dm who performed a high level of physical activity did not differ from controls in hrv ; however , children with t1 dm who had low level of physical activity had significantly reduced hrv compared to both active children with t1 dm and non - t1 dm children . thus , the current study provides support that exercise can be an effective means to improve hrv in t1 dm . both tachycardia and bradycardia responses were studied in the context of brs analysis in order to explore the control features related to unloading or loading of the baroreceptors , respectively . some discrepancy exists between different experimental models of t1 dm and their impact on brs measures . investigations using the hyperglycemic non - obese diabetic ( nod ) t1 dm mouse model have shown elevations in brs measures rather than attenuated responses . in contrast , tachycardic - snp and bradycardic - pe responses have been shown to be lower in stz - induced t1 dm hyperglycemic rats in comparison to non - t1 dm controls but were improved with exercise training . in the current study , the slope of the hypotensive tachycardia response was not significantly different between any of the groups suggesting that responses to baroreceptor unloading are not affected by t1 dm or exercise . however , t1 dm reduced the bradycardia response to baroreceptor loading , which was nullified by concurrent exercise training . these findings are in line with previous reports demonstrating a bradycardia change in pe - brs without an accompanying change in snp - brs , which was improved following aerobic exercise . discrepancies in brs responses in t1 dm models seem to be closely associated with both the duration and the severity of diabetes . a recent study examining the time - course of brs changes in response to stz - induced hyperglycemia reported that alteration of the snp - brs was not evident until 12 weeks of diabetes , while a change in pe - brs was evident as early as 4 weeks after induction . interestingly , the animals in the aforementioned study were moderately hyperglycemic ( 1618 mm ) , suggesting that the severity of the hyperglycemia may play a role in the progression of this neuropathy . vinik and ziegler ( 2007 ) reported that poor glycemic control and duration of diabetes play a central role in progression of cardiovascular autonomic neuropathy . yet , it is not clear what role insulin therapy may play in the neuropathy . insulin supplementation to stz - induced t1 dm rats can modify the changes in brs sensitivity evident at 48 weeks of t1 dm . indeed , in clinical t1 dm patients , intensive therapy is well documented to slow the progression and delay the appearance of abnormal autonomic function . however , the current study provides evidence that the ability of exercise to ameliorate cardiovascular autonomic dysfunction may be independent of its ability to reduce [ bg ] , which challenges the direct relationship between [ bg ] and can suggested by previous studies [ 6769 ] . the ivgtt performed at the conclusion of the 10-week exercise period demonstrated an increased glucose clearance rate ( kg ) and therefore glucose tolerance , in the cx group compared to the preexercise training period . however , in both the sedentary and exercise diabetic groups there was an equal decline in kg to nearly the same rate . this decrease was significantly different from pre - t1 dm values and the week 10 values of the c and cx groups . while this would normally indicate that both of the diabetic groups developed equally impaired glucose tolerance , it was also the case that the dx group required approximately half of the dosage of exogenous insulin compared to the d group to maintain their [ bg ] in the 917 mm range . with double the insulin dose , it is likely that the total serum insulin over a given time during ivgtt would have been greater in the d than dx group , and with their kg being equal , that would indicate that there was a greater insulin sensitivity in the diabetic exercise group [ 70 , 71 ] . together , these ivgtt results demonstrate that exercise training improved glucose tolerance and insulin sensitivity . furthermore , since the [ bg ] of the diabetic groups in this study was held in a constant range , any abovementioned exercise - induced improvements to cv autonomic function would not have been mediated through a reduction in systemic [ bg ] but may have been the result of improvements in insulin sensitivity and glucose utilization [ 72 , 73 ] . this should be borne in mind when considering the effects of diabetes and exercise on indices of cv autonomic function , such as hrv and brs . an alternative mechanism by which exercise can influence brs was reported by bernardi et al . they demonstrated that a reduced parasympathetic brs in patients with t1 dm was improved by both oxygen supplementation and deep breathing to the same degree , which indicated the increased respiration and oxygen delivery resultant of exercise could have been mediating increases in brs . this led the authors to suggest that hypoxia in t1 dm functionally restrains parasympathetic activity . however , reduced brs could also be attributed to defects in the baroreceptors , baroreceptor afferent nerves , cns structures , or efferent fibres of the baroreflex circuit [ 7 , 8 , 61 ] . in the present study , the finding that the tachycardia response of the baroreflex was unimpaired by t1 dm , while the bradycardia response was , suggests that the afferent arm and central regulators of the baroreflex were not dysfunctional and that the observed decrement of baroreflex bradycardia may have been caused partly by alterations in efferent parasympathetic outflow [ 8 , 29 ] . another interesting outcome of the current study was the alteration of sympathetic vasomotor control in the d group , which was also modified by concurrent exercise training . in this study , prazosin treatment resulted in a drop in map that was approximately twofold greater in the d group compared to the c and dx groups , which is indicative of a much greater sympathetic contribution to the maintenance of baseline vascular resistance [ 75 , 76 ] . similarly , martinez - nieves and dunbar ( 1999 ) reported that male t1 dm rats had a greater decrease in map after a bolus injection of prazosin compared to their control cohorts . however , they postulated that an elevated prazosin response could be the result of increased 1-adrenergic receptor sensitivity . yet , in this study , the finding that treatment with pe , an 1-adrenergic receptor agonist , did not result in a greater peak sbp , nor a greater percent increase in sbp from baseline in the t1 dm group ( data not shown ) , argues against a receptor - based sensitivity mechanism and , rather , suggests that efferent sympathetic outflow may have been elevated in the d group . however , we can not determine the mechanism that resulted in prazosin showing a preferential decrease in map in the d group versus dx or c based on the data in this study . yet , in line with the current results , such elevations in resting sympathetic activity would make activation of the brs response to snp - induced hypotension more difficult . the conclusion above regarding sympathetic hyperactivity in the d group is supported by measurements of neuropeptide y [ npy ] obtained in this study . [ npy ] is coreleased with norepinephrine from perivascular and cardiac sympathetic nerve terminals during sympathetic activation [ 78 , 79 ] . in clinical t1 dm , a diabetes - related decrease in [ npy ] is attributed to impaired sympathetic function , whereas increased [ npy ] is attributed to sympathetic overactivity [ 7981 ] . in the current study , serum [ npy ] was greater in both of the t1 dm groups in comparison to their control groups . thus , despite the ability of exercise to preserve reflex cardiac function in t1 dm , hyperglycemia itself appears to have impacted basal vascular adrenergic activity in both t1 dm groups . as both t1 dm groups were maintained at equally elevated [ bg ] , there may have been a correspondingly similar stimulation of peripheral sympathetic activation and npy release [ 79 , 82 , 83 ] . despite improvements by exercise training to deficits of cardiovascular autonomic function , no observable statistical differences in either map or hr were evident between any of the groups . indeed , it has been shown that alterations in autonomic function occur before or without alterations in map and hr and are uncorrelated to changes in sympathetic tone . the observed changes in basal sympathetic activity may assist in the maintenance of blood pressure , ventricular function , and cardiac output during the early stage of diabetes , which is supported by our findings that inhibition of sympathetic activity results in a greater decrease in map in diabetic rats than normal rats . in that respect , we previously reported that although t1 dm animals demonstrated significant alterations in myocardial dimensions and structure , measurements of cardiac performance ( ejection fraction , fractional shortening , and cardiac output measurements ) were unchanged . to evaluate the heart rate of these animals without neural influence , we measured the beat rate of denervated hearts using the isolated langendorff technique . we found that the ihr of the d group was lower than both c and dx groups , which would support the notion that decreased ihr masked the effects of sympathetic overactivity in the current study . further , it supports evidence that stz - induced diabetes may have a direct effect on heart rate by modifying the heart itself [ 86 , 87 ] . interestingly , in some studies , insulin therapy was only able to partially reverse bradycardia and it was shown that stz - treatment itself could lengthen the action potential duration in the sa node , slowing the hr . however , if hyperglycemia or stz directly affected cardiac muscle or the sa node and caused a decreased ihr in the d group , it is also the case that exercise training rescued or prevented the deficit , as the ihr of the dx group was not different from the cx group . thus , previous experimental t1 dm studies that reported that stz - induced bradycardia and hypotension were caused by can , and that exercise - induced normalization of hr and bp was evidence of improvements in autonomic function , may really have been observing changes in intrinsic cardiac function which were independent of autonomic control . such changes could instead have been due to depressed sarcoplasmic reticulum function or impaired calcium handling [ 87 , 89 , 90 ] . therefore , the direct effects of stz on the heart and ihr require further examination and should be taken into consideration in future studies that investigate the autonomic regulation of cv function in stz - induced t1 dm models . an important consideration regarding the design of the current study was the use of anesthetized rats . in order to accurately reflect cardiovascular parameters in such a state , we selected an anaesthetic regime that provides the lowest level of influence on baseline and reflexive cv control attainable in rodent models . a light plane anesthesia 0.51.2 g / kg has been shown to maintain the integrity of the cardiovascular system , where higher doses of urethane ( above 1.5 g / kg ) can produce hypotension and bradycardia , as well as high rates of mortality [ 91 , 92 ] . in the current study we used a minimal dose of 25 mg / kg , which was reported in previous studies by our laboratory to have little influence on neurovascular blood flow measures [ 9395 ] . that being said , it can not be determined to what extent , if at all , the autonomic nervous system was augmented by the urethane - chloralose treatment in comparison to conscious animals . further work examining a comparison of our anesthesia regime with freely moving conscious animals ( using telemetry devices ) will better address this matter . in this study , t1 dm induced indications of parasympathetic withdrawal , sympathetic overactivity , and , despite a decreased ihr , no change in resting map or hr . however , concurrent exercise training with t1 dm maintained the sensitivity of the parasympathetically mediated baroreflex bradycardia , prevented an increase in vascular sympathetic tone , maintained a higher bodyweight , and prevented a decrease in ihr . the ability of exercise training to preserve parasympathetic function in this model of t1 dm indicates that the exercise - mediated improvements to parasympathetic function are independent of alterations in [ bg ] . however , the finding that [ npy ] remained elevated suggests that hyperglycemia has a direct impact on adrenergic activity . taken together , our t1 dm model of progressive stz induction and insulin treatment induced autonomic impairments similar to those observed in clinical t1 dm and demonstrates the novelty of this model for investigating the effectiveness of high intensity aerobic exercise training as a means to prevent the progression of can in t1 dm . thus , although not examined in this study , the mechanisms that underlie the physiological changes caused by t1 dm and exercise can be the focus of future investigations using this model .

indices of cardiovascular autonomic neuropathy ( can ) in experimental models of type 1 diabetes mellitus ( t1 dm ) are often contrary to clinical data . here , we investigated whether a relatable insulin - treated model of t1 dm would induce deficits in cardiovascular ( cv ) autonomic function more reflective of clinical results and if exercise training could prevent those deficits . sixty - four rats were divided into four groups : sedentary control ( c ) , sedentary t1 dm ( d ) , control exercise ( cx ) , or t1 dm exercise ( dx ) . diabetes was induced via multiple low - dose injections of streptozotocin and blood glucose was maintained at moderate hyperglycemia ( 917 mm ) through insulin supplementation . exercise training consisted of daily treadmill running for 10 weeks . compared to c , d had blunted baroreflex sensitivity , increased vascular sympathetic tone , increased serum neuropeptide y ( npy ) , and decreased intrinsic heart rate . in contrast , dx differed from d in all measures of can ( except npy ) , including heart rate variability . these findings demonstrate that this t1 dm model elicits deficits and exercise - mediated improvements to cv autonomic function which are reflective of clinical t1 dm .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

PMC4085201

oxidative stress refers to an imbalance between free radical [ especially reactive oxygen species ( ros ) ] generation and antioxidant defense1 . the antioxidant system in the human body includes enzymatic [ e.g. superoxide dismutase ( sod ) and glutathione peroxidase ( gpx ) ] and non - enzymatic antioxidants [ e.g. glutathione ( gsh ) ] and it plays an important role in the prevention of oxidative stress . oxidative stress is involved in the pathogenesis of hypertension , atherosclerosis , diabetes , osteoporosis , and cancer2 . ros initiates lipid peroxidation through an attack on polyunsaturated fatty acids , and generates products such as mda ( malondialdehyde)4 . ros also causes oxidative damage to proteins and dnas4 . among these damages , oxidative dna damage is the most detrimental one to human health because of its role in the pathogenesis of the various diseases mentioned above . ros , especially hydroxyl radical ( oh ) , can cause dna base changes , strand breaks , damage to tumor - suppressor genes , and enhanced expression of proto - oncogenes5 . in addition to the increase of ros , the decrease of dna repair capacity is the other reason for the accumulation of oxidative dna damage in the human body6 . physical exercise is an important factor affecting oxidative stress and oxidative dna damage , since a sharp increase in oxygen consumption during exercise results in an increase in ros generation . however , the effect of exercise - induced oxidative damage is variable depending on several factors such as type , mode , duration , and intensity of exercise7 . a single bout of exercise induces oxidative stress and dna damage , whereas regular moderate intensity exercise decreases them8 . under the concept of hormesis , low - to - moderate ros generation induced by regular moderate intensity exercise a previous study demonstrated that long - term high - intensity exercise ( 75%vo2max ) increased 8-hydroxy-2-deoxyguanosine ( 8-ohdg ) , while regular exercise with moderate - intensity ( 50%vo2max ) tended to decrease 8-ohdg10 . the activity of 8-oxoguanine dna glycosylase ( ogg1 ) , a crucial enzyme for the repair of 8-oxoguanine lesions , is up - regulated after regular exercise in human and animals11 . tai chi ( tc ) , a traditional chinese exercise , is classified as a moderate intensity exercise , and its intensity is basically similar among different age and gender groups12 . previous studies have reported the numerous tangible benefits of tc practice , such as improvement of balance and prevention of falls , enhancement of flexibility and strength , as well as amelioration of cardio - respiratory and immune function impairments . studies of the effects of tc exercise on oxidative stress and oxidative dna damage are limited . tc exercise reduced the degree of oxidative stress in middle - aged women13 , older adults14 , and obese patients with type 2 diabetes15 , and long - term tc practice reduced the degree of dna damage , measured by the comet assay , in middle - aged13 and older16 adults . however , the effects of tc intervention on oxidative stress and dna damage have not been investigated in young females who seldom participate in physical exercise . the efficacy of tc exercise on dna base damage and its repair also remains unclear . young females are less conscious about physical exercise compared to age - matched males17 . although tc exercise is suitable for any population18 , only middle - aged and elderly age groups frequently practice tc for health purposes . tc is becoming popular among chinese college students since it has been designated as a compulsory sport in the physical education curriculum of most colleges . therefore , the objective of this pilot study was to determine the effects of tc intervention on oxidative stress , dna damage and repair in young females who did not participate in regular physical exercise . ten sedentary female students from gannan normal university , china , voluntarily participated in this program . they completed inclusion and lifestyle questionnaires . exclusion criteria were as follows : having experience of tc exercise ; having performed physical exercise for more than 1 hr per week in the past 3 months ; smoker or regular alcohol drinker ; consuming supplements or medical products with antioxidant properties ; having a history of cardiovascular and respiratory diseases ; having knee joint pain ; having a history of lower - extremity fracture within the past 1 year . they were asked to keep their usual lifestyle including dietary behavior and daily activities , and medication ( if any ) throughout the study period . at the time of recruitment , their demographic characteristics were as follows : age , 20.400.70 yrs old ; height , 155.486.15 cm ; weight , 47.136.11 kg ; body mass index ( bmi ) , 19.421.54 kg / m ; and resting heart rate , 79.1012.65 beats / minute . all of the participants completed the 12-week tc training program . according to this protocol , they practiced the 24-form simplified tc , 5 times ( sessions ) per week ( monday to friday ) , on campus . the first 2 weeks involved familiarization with tc through teaching and learning . in the following 10 weeks , the participants practiced 5 rounds of tc ( 1 round refers to practice from the 1st movement to the 24th movement of this tc style ) accompanied by a classic music for this tc style in each session . an experienced tc instructor led all participants in practice and taught them necessary movement corrections in the intervals between rounds . thus , each session lasted about 60 min including a 10-minute warm - up for stretching and a 5-minute cool - down for relaxation . heart rate ( hr ) during tc exercise was monitored in the last session of the 5 rounds of the 24-form simplified tc in the last 4th , 8th , and 12th weeks . the exercise duration of five rounds of tc practice lasted for around 30 minutes . hr parameters such as average hr , maximum hr , and minimum hr were recorded using a polar watch ( suunto t6d running pack , finland ) and the telemetry hr team system ( suunto team manager , finland ) . blood samples were collected at baseline , 8 , and 12 weeks . in each session of blood collection , 3 ml of whole blood was taken after overnight fasting between 7:308:30 a.m. from a forearm vein and placed into a heparinized tube . plasma was separated by centrifugation at 3,000 rpm for 5 min and kept frozen at 80c until assayed for 8-ohdg , ogg1 , sod , gpx , mda , gsh , and hydroxyl radical inhibiting capacity ( oh-ic ) . sod was measured by the hydroxylamine assay using a spectrophotometer ( unico7200 , usa ) at 550 nm . gpx was detected by the rate method using a spectrophotometer ( unico7200 ) at 412 nm . mda was assessed by the thiobarbituric acid ( tba ) assay using a spectrophotometer ( unico7200 ) at 532 nm . gsh was analyzed by colorimetry using a spectrophotometer ( unico7200 ) at 420 nm . hydroxyl radical inhibiting capacity was determined by the griess colorimetric method using a spectrophotometer ( unico7200 ) at 550 nm . the kits for sod , gpx , mda , gsh , and oh-ic measurements were all purchased from nanjing jiancheng bioengineering institute , china . plasma 8-ohdg and ogg1 levels were measured using commercial elisa ( enzyme - linked immunosorbent assay ) kits according to the manufacturer s instructions ( santa cruz biotechnology , inc . then , either 10 l of anti-8-ohdg or anti - ogg1 antibody in combination with 50 l of streptavidin - biotin - horseradish peroxidase were added to the sample wells in turn . to the standard sample wells , 50 l of 5 different concentrations of standard samples and 50 l of streptavidin - biotin - horseradish peroxidase were added . the microtiter plate was then incubated for 1 hour at 37 c . then , the plate was washed with wash buffer 5 times . during the procedure of washing , before discarding the buffer , each well was filled with wash buffer for 30 seconds . color reactions were then conducted for 10 min in the dark at 37 c after 50 l of chromogenic agent a and 50 l of chromogenic agent b were added in turn to each well . finally , 50 l of stop solution was added to terminate the reaction and the blue color changed to yellow . shanghai , china ) at 450 nm within 15 min , and the concentrations of 8-ohdg and ogg1 in plasma were read from standard curves . all data were expressed as the mean sd . the differences of plasma and hr monitoring parameters among different time points were determined by repeated measures analysis of variance ( anova ) . post hoc pair - hoc comparisons ( lsd ) were performed to test the differences when significance was shown . the correlation between plasma 8-ohdg and ogg1 levels at the same time point was determined using pearson s correlation coefficient . the statistical package for social sciences ( spss ) version 17.0 was used for the analysis and values of p<0.05 were considered significant . when the participants were performing 5 rounds of tc , the average hr and maximum hr tended to increase at 4 , 8 , and 12 weeks , but they did not show significant differences ( table 1table 1 . comparison of hr among the different time pointsvariables4 weeks8 weeks12 weekshraverage ( bmp)108.310.5110.0010.7113.918.8hrmax ( bmp)124.212.4126.913.4132.922.5hrmin ( bmp)88.310.987.78.384.810.4hraverage = mean heart rate , hrmin = minimum heart rate , hrmax = maximum heart rate , bmp = beats per minute ) . in contrast , the minimum hr tended to decrease during this study period , although the decreases were not statistically significant ( p>0.05 ) ( table 1 ) . hraverage = mean heart rate , hrmin = minimum heart rate , hrmax = maximum heart rate , bmp = beats per minute the sod level was significantly increased ( p<0.05 ) at 8 and 12 weeks of tc exercise compared to the baseline value ( table 2table 2 . effects of tai chi exercise on oxidative stress markersvariablesbaseline8 weeks12 weekssod ( u / ml)42.763.0549.815.01 * 49.054.73*gpx ( u / ml)130.3022.88133.5318.77141.6028.55mda ( nmol / ml)2.770.412.120.41 * 2.970.54gsh ( mggsh / l)11.925.0710.483.089.194.56oh-ic ( u / ml)351.9759.02406.1934.76 * 408.4949.86**p<0.05 compared to baseline , sod : superoxide dismutase ; gpx : glutathione peroxidase ; mda : malondialdehyde ; gsh : glutathione ; oh-ic : hydroxyl radical inhibiting capacity ) . conversely , the plasma mda level was significantly decreased at 8 weeks but not at 12 weeks compared to the baseline ( table 2 ) . however , gpx and gsh levels did not alter significantly during the study period . * p<0.05 compared to baseline , sod : superoxide dismutase ; gpx : glutathione peroxidase ; mda : malondialdehyde ; gsh : glutathione ; oh-ic : hydroxyl radical inhibiting capacity although the increases were not statistically significant , the plasma 8-ohdg values in this study showed increases at 8 and 12 weeks compared to the baseline value ( table 3table 3 . comparison of plasma 8-ohdg and ogg1 , and their relationships at different time pointsvariablesbaseline8 weeks12 weeks8-ohdg ( ng / ml)9.452.5410.932.5110.262.34ogg1 ( ng / ml)6.912.058.171.54 * 8.491.62*r ( p value)0.842 ( 0.003)0.78 ( 0.008)0.545 ( 0.103)*p<0.05 compared to baseline , 8-ohdg : 8-hydroxy-2-deoxyguanosine ; ogg1 : 8-oxoguanine dna glycosylase ; r : correlation coefficients of plasma 8-ohdg and ogg1 at the same time point ) . in contrast , the plasma ogg1 level was significantly increased at 8 and 12 weeks compared to the baseline value ( table 3 ) . positive correlations between plasma 8-ohdg and ogg1 were found at baseline ( p=0.003 ) and 8 weeks ( p=0.008 ) , but no statistically significant correlation ( p=0.103 ) was found at 12 weeks ( table 3 ) . * p<0.05 compared to baseline , 8-ohdg : 8-hydroxy-2-deoxyguanosine ; ogg1 : 8-oxoguanine dna glycosylase ; r : correlation coefficients of plasma 8-ohdg and ogg1 at the same time point several empirical studies using different parameters , including heart rate ( hr ) , oxygen consumption ( vo2 ) , and energy expenditure , have shown that tc is an aerobic exercise of moderate intensity12 . however , in those previous studies , the intensity of tc was measured only when participants practiced one round of tc , from the first movement to the last one of the specific tc style . it is intuitive that several factors such as the individual variances in age , gender , proficiency levels of tc , and also tc style would affect the intensity . in the present study , we monitored the participants hr when they practiced 5 rounds of the 24-form simplified tc at 4 , 8 , and 12 weeks . the results of hr monitoring show that the average hr during tc practice corresponded to 54.3% , 55.1% , and 57.1% of the age - predicted maximum heart rate ( = 220-age ) at 4 , 8 , and 12 weeks , respectively . these results indicate that the intensity of tc practice of the present protocol was moderate for the young female students . although exercise has numerous benefits for human health , it is also often claimed to induce oxidative stress since exercise increases ros generation . ros generated during exercise is , however , not always harmful because moderate and gradual exercise helps people to acquire antioxidant defenses19 . the antioxidant defense system in the human body includes enzymes , such as sod and gpx , and non - enzymatic antioxidants such as gsh . in the enzymatic system , sod is the first defense against oxidative stress and is the major defense against o2 , whereas gpx is responsible for scavenging h2o2 and other organic peroxides . , we observed a significant increase in plasma sod and oh-ic levels after 8 weeks of tc exercise indicating that 8 weeks of tc exercise following our protocol is sufficient enough to improve the scavenging capacities for o2 and oh. in this study , the plasma mda level decreased at 8 weeks . thus , tc exercise can help to reduce the lipid peroxidation products attacked by ros . in the present study , although the increase was not statistically significant , gpx tended to increase with time of tc practice . since gpx is efficiently induced by high ros concentrations20 , our protocol would have generated low concentrations of ros . our results are in agreement with those of previous studies concerning the protective effects of tc exercise on oxidative stress in middle - aged and elderly subjects13 , 14 . we propose there are two possible mechanisms of regular moderate - intensity exercise which reduce the markers of oxidative stress . first , based on the theory of hormesis , low - to - moderate oxidants would cause up - regulation of antioxidant enzymes9 . ros generated in this type of exercise are not only toxic , but also play an important role in cell signaling and in the regulation of gene expression9 , 19 . in this sense , second , the cumulative effects of repeated exercise bouts and exposure to ros during regular moderate - intensity exercise could be the other mechanism19 . in addition to the changes of oxidative stress markers , no significant changes in plasma 8-ohdg were observed throughout this study . in attack by ros ( especially oh ) , 8-ohdg is the most ubiquitous product of oxidative dna base modification which occurs in approximately 1 in 100,000 guanidine residues in a normal human cell22 . for example , the plasma 8-ohdg level was increased by 12 weeks of high - intensity exercise , whereas it tended to decrease after moderate - intensity exercise in young men10 . 8-ohdg was significantly lower in a moderate endurance exercise group than in a sedentary group4 . after 10 months of aerobic exercise , the 8-ohdg level was increased significantly compared to the baseline in a group of elderly subjects23 . inconsistency among these outcomes may be due to the different age groups , exercise types and modes , as well as the exercise intervention duration . an animal study also showed that 2 months of regular treadmill running significantly upregulated the activity of ogg1 in hepatocyte nuclei of old rats compared to a sedentary old group11 . another animal study showed that exercise training increased ogg1 levels / activity in the nucleus of skeletal muscles and specific activity of ogg1 in mitochondrial compartments26 . similarly , our present study demonstrated that the plasma ogg1 level significantly increased after tc practice for 8 and 12 weeks . increase of this enzyme in plasma is likely to be an adaptive response to the increased oxidative damage induced by elevated ros during tc exercise . the damaged dna with 8-ohdg may contribute to point mutation during the subsequent replication27 . with normal body repair mechanisms , 8-ohdg can be cleaved by ogg1 through the way of base excision pathway and normal guanine is added to the site24 . for example , kondo et al . showed a positive association between 8-ohdg levels and ogg1 expression ( r=0.702 , p<0.05)28 . in our study , significant positive correlations between plasma 8-ohdg and ogg1 were observed at baseline ( r=0.824 , p<0.01 ) and 8 weeks ( r=0.78 , p<0.01 ) ; however , no significant correlation was found at 12 weeks ( r=0.545 , p>0.05 ) . these results suggest that the increase of ogg1 activity after 12 weeks of tc exercise exceeds the requirements for immediately excising all 8-ohdg in young females . based on the results of this pilot study , a 12-week tc exercise intervention is effective at increasing the activity of plasma sod and ogg1 and decreasing plasma mda concentration in young sedentary females . it suggests that tc exercise helps to alleviate the oxidative stress response and dna damage in those who have serious levels of oxidative stress and dna damage , such as older and sick people . third , the authors only told the participants to keep their daily dietary habits and did not ask them to record their dietary details , especially before each blood sampling . thus , dietary variation might have affected the data of blood parameters . nevertheless , this pilot study showed tc intervention can improve the response to oxidative stress and the dna damage / repair process in female students . further studies with a longer intervention duration , larger groups , and a control group are needed to confirm our present results . moreover , the long - term effects of tai chi on oxidative stress response and dna / repair also need to be studied in the future .

[ purpose ] this study was to examine the effects of 12 weeks of tai chi ( tc ) exercise on antioxidant capacity , and dna damage / repair in young females who did not perform regular physical exercise . [ subjects and methods ] ten female students from a chinese university voluntarily participated in this program . all of them practiced the 24-form simplified tai chi , 5 times weekly , for 12 weeks . plasma levels of superoxide dismutase ( sod ) , glutathione peroxidase ( gpx ) , malondialdehyde ( mda ) , glutathione ( gsh ) , hydroxyl radical inhibiting capacity ( oh-ic ) , 8-hydroxy-2-deoxyguanosine ( 8-ohdg ) , and 8-oxoguanine dna glycosylase ( ogg1 ) were measured at 0 , 8 , and 12 weeks . heart rate ( hr ) was monitored during the last set of the training session at 4 , 8 , and 12 weeks . [ results ] plasma sod and oh-ic levels were increased at 8 and 12 weeks compared to the baseline ( 0 weeks ) . gpx and gsh levels did not change significantly throughout the study period . the plasma mda level was decreased significantly at 8 weeks but not at 12 weeks compared to the baseline value . while the plasma 8-ohdg level did not change throughout the study period , the plasma ogg1 level was significantly increased at 8 and 12 weeks compared to the baseline value . [ conclusion ] tc practice for 12 weeks efficiently improved the oxidative stress response in young females who did not perform regular physical exercise . the tc exercise also increased the dna repairing capacity .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC4550771

matrix metalloproteinases ( mmps ) are zn - dependent endopeptidases that are implicated in numerous physiological processes such as reproduction , wound repair , development , and immune function as well as pathological conditions such as cancer , inflammation , neurodegenerative disorders , and autoimmune diseases [ 14 ] . the expression of many mmp family members is enhanced in endotoxemia [ 57 ] and cecal ligation and puncture ( clp ) [ 810 ] , two mouse models of sepsis , a highly mortal infectious disease , which still lacks an efficient therapeutic treatment . additionally , studies using mmp inhibitors and mmp knockout mice indicate that mmps play a detrimental role in sepsis [ 9 , 1113 ] . moreover , several human data have directly correlated higher levels of active mmp with lower rates of survival in sepsis patients and some mmps are considered as valuable biomarkers in sepsis [ 8 , 9 , 14 , 15 ] . their detrimental role in several inflammatory diseases justifies the development of specific mmp inhibitors , which have been intensely sought after for over two decades but with poor success [ 4 , 16 ] . most mmp inhibitors ( mmpi ) consist of two distinct features : a peptidomimetic backbone that interacts noncovalently with specific subsites of the catalytic domain that govern substrate interaction and a zn - chelating moiety or zinc - binding group ( zbg ) that binds to the hydrolytic zn - ion via coordinate - covalent bonds , rendering the enzyme inactive [ 16 , 17 ] . the majority of mmpi were initially synthesized containing hydroxamic acid as zbg which , although very potent , resulted in poor specificity , oral availability , or biocompatibility . to circumvent these problems , pyrone - based inhibitors emerged as superior mmpi with known biocompatibility , good aqueous solubility , and synthetic versatility [ 19 , 20 ] . based on this strategy , a specific mmp-3 pyrone - based inhibitor ( am-6 ) was developed . however , the specificity of am-6 was only characterized for mmp-1 , mmp-2 , and mmp-3 and therefore , in this study , we synthesized the same compound and screened it against a broader range of mmps to firmly ascertain mmp selectivity . surprisingly , the compound displayed specificity and potency towards mmp-12 . to accommodate these findings , we show that the inhibitor protects against inflammation - induced disruption of the blood - cerebrospinal fluid barrier both in vitro and in vivo . the target compound was synthesized according to the literature with some modifications ( scheme 1 ) . the omission of an aqueous recrystallization step leads to near - quantitative yields of the hydrolytically unstable chlorokojic acid ( 1 ) without sacrificing purity . furthermore , 4-iodobenzylamine could be replaced with 4-bromobenzylamine ( g ) , which is less costly and not sensitive towards light and air . the hcl / acetic acid protocol as written in the original publication did not provide us with the final inhibitor in good yield ( some 39% crude yield ) . reductive debenzylation ( step i ) did prove to be a very reliable method to obtain the deprotected molecule . proton nmr spectra were recorded using a bruker avance 300 ( 300 mhz ) device using tetramethylsilane as internal standard for cdcl3 and dmso - d6 . c - nmr spectra were recorded using a bruker avance 300 ( 75 mhz ) device , using the deuterated solvent as an internal standard . reagents were purchased from various companies , most notably tci , sigma - aldrich , and acros . kojic acid ( tci , 15 g , 0.105 mol ) is placed in a round bottom flask equipped with a magnetic stirring bar . the flask is cooled in an ice bath and thionyl chloride ( 60 ml ) is slowly added after which the ice bath is removed and the mixture is placed under an argon atmosphere . after stirring for 1.5 h a yellow mass is filtered off and washed extensively with petroleum ether . the product was an off - white solid ( 16.6 g , 98% ) : h - nmr ( dmso - d6 ) 4.6 ( s , 2h , cl - ch2 - 6 ) , 6.6 ( s , 1h , 5-h ) , and 8.1 ( s , 1h , 2-h- ) in agreement with the literature . chlorokojic acid 1 ( 2.5 g , 0.0156 mol , 1 eq ) is dissolved in water ( 8 ml ) and heated to 50c under an argon atmosphere in a flask equipped with a magnetic stirring bar . zinc dust ( 2.1 g , 0.031 mol , 2 eq ) is added . an aqueous solution of hcl ( 4.7 ml , 0.055 mol , 3.5 eq ) is slowly added to the reaction mixture under vigorous stirring , during which the temperature is increased to 70c . after 4 h the excess zinc dust is filtered off while being hot , and the resultant liquid is extracted with dichloromethane ( 320 ml ) . the combined organic phase is dried over magnesium sulfate and the solvent is removed by evaporation under reduced pressure . the crude material is purified via recrystallization from isopropanol to obtain an off - white solid ( 1.2 g ; 52% ) : h - nmr ( cdcl3 ) 2.3 ( s , 3h ) , 6.2 ( s , 1h ) , and 7.8 ( s , 1h ) in agreement with the literature . sodium hydroxide ( 0.696 g , 0.017 mol , 1.1 eq ) is dissolved in water ( 16 ml ) . to this solution 2 ( 2 g , 0.016 mol , 1 eq ) a magnetic stirring bar is added . a 35% aqueous formaldehyde solution ( 0.36 ml , 0.017 mol , 1.1 eq ) is added and the mixture is allowed to stir overnight . after acidification to ph 1 with 35% aqueous hydrochloric acid the mixture is cooled to 2c and allowed to stand overnight . the crystalline mass is filtered off , washed with 5 ml of water , and dried via lyophilisation . an off - white solid is obtained ( 1.71 g , 71% ) : h - nmr ( dmso - d6 ) 2.26 ( s , 3h ) , 4.38 ( s , 2h ) , 5.36 ( b , s , 1h ) , 6.21 ( s , 1h ) , and 8.86 ( b , s , 1h ) in agreement with the literature . sodium hydroxide ( 0.56 g , 8.93 mmol , 1.1 eq ) is dissolved in water ( 2.4 ml ) and added to a solution of 3 ( 2 g , 8.12 mmol , 1 eq ) in methanol ( 1.2 ml ) in a two - necked flask under argon equipped with a reflux condenser , a septum , and a magnetic stirring bar . benzyl bromide ( 1.7 ml , 8.93 mmol , 1.1 eq ) is slowly added and the mixture is allowed to stir overnight . the mixture is concentrated in vacuo and taken up in dichloromethane ( approximately 20 ml ) . is then extracted with 5% aqueous sodium hydroxide ( 220 ml ) and brine ( 120 ml ) . the organic phase is dried with anhydrous sodium sulfate after which the solvent is removed under reduced pressure . the solid is dissolved in as little dichloromethane as possible after which heptane ( or petroleum ether ) is added until no more precipitation occurs . the precipitate is filtered off and dried to yield a white to off - white powdery solid ( 2.3 g , 73% ) : h - nmr ( cdcl3 ) 2.25 ( s , 3 ) , 2.284.30 ( d , 2h , 6.8 hz ) , 5.18 ( s , 2h ) , 6.19 ( s , 1h ) , and 7.37 ( s , 5h ) in agreement with the literature . compound 4 ( 2 g , 1 eq , 8.18 mmol ) is dissolved in 37.8 ml chloroform and 10.2 ml dmso . to this mixture triethylamine ( 7 ml , 0.050 mol , 6 eq ) and pyridine ( 0.1 ml ) are added . the mixture is stirred in an ice bath under argon using a magnetic stirring bar until a temperature of 35c is reached . sulfur trioxide - pyridine complex ( 6.48 g , 0.040 mol , 5 eq ) is subsequently added and the mixture is allowed to stir overnight . it is then extracted ( 220 ml water , 120 ml brine ) and dried over anhydrous sodium sulfate . the solvent is removed under reduced pressure using a rotavapor equipped with a cold trap ( dry ice / isopropanol ) to obtain the crude product as orange oil . this is diluted with 10 ml diethyl ether and transferred onto a flash column packed with silica / diethyl ether . after eluting with diethyl ether the solvent is removed under reduced pressure using a rotavapor equipped with a cold trap . a white to yellow crystalline solid is obtained ( 1.38 g , 69% ) : h - nmr ( cdcl3 ) 2.32 ( s , 3h ) , 5.49 ( s , 2h ) , 6.31 ( s , 1h ) , 7.35 ( s , 5h ) , and 9.85 ( s , 1h ) in agreement with the literature ; ft - ir 1690.33 cm ( c = o aldehyde ) , 1641.24 cm ( c = o pyrone ) , 1613.12 cm , and 1583.88 cm ( c = c pyrone ) . ( 6 ) preparation of compound 6 ( 3-(benzyloxy)-6-methyl-4-oxo-4h - pyran-2-carboxylic acid ) . compound 5 ( 1.57 g , 6.4 mmol , 1 eq ) is dissolved in acetone / water ( 20 ml/20 ml ) with a magnetic stirring bar . sulfamic acid ( 0.86 g , 8.9 mmol , 1.4 eq ) and sodium chlorite ( 80% , 0.76 g , 6.7 mmol , 1.05 eq ) are added and the mixture is allowed to stir for 1 h in an open vessel . the mixture is subsequently evaporated under reduced pressure to remove the acetone after which the crude product is filtered off and washed with a small amount of ethanol . the product is then dried in vacuo to obtain a stark white powder ( 1.3 g , 78% ) : h - nmr ( dmso - d6 ) 2.29 ( s , 3h ) , 5.10 ( s , 2h ) , 6.40 ( s , 1h ) , and 7.327.45 ( m , 5h ) in agreement with the literature ; ft - ir 1720.37 cm ( c = o acid ) , 1625.50 cm ( c = o pyrone ) , 1554.48 cm and 1496.36 cm ( c = c pyrone ) , and mp ( precipitated from water / acetone ) 180c . compound 6 ( 0.1 g , 0.38 mmol , 1 eq ) is dissolved in dry tetrahydrofuran ( 4.16 ml ) in a flame - dried round bottom flask equipped with a magnetic stirring bar under an argon atmosphere . n - hydroxysuccinimide ( 0.045 g , 0.39 mmol , 1.02 eq ) is added and the mixture stirred for 30 min . dicyclohexylcarbodiimide ( 0.079 g , 0.38 mmol , 1 eq ) is added as a solid and the mixture is stirred for an additional 3 h at rt under an argon atmosphere . dicyclohexylurea is filtered off and the precipitate is washed with tetrahydrofuran ( 2 ml ) . 4-bromobenzylamine ( 0.05 ml , 0.385 mmol , 1.01 eq ) is added to the filtrate in a dried round bottom flask equipped with a magnetic stirring bar and a reflux condenser under an argon atmosphere . this solution is transferred to a chromatography column ( silica , eluent chloroform / methanol 1% ) . after chromatography , the product is obtained as an off - white solid ( 0.15 g , 91% ) : h - nmr ( cdcl3 ) 2.37 ( s , 3h ) , 4.34.38 ( d , 2h , j = 5.6 ) , 5.33 ( s , 2h ) , 6.28 ( s , 1h ) , 6.97.5 ( m , 9h ) , and 8.06 ( b , s , 1h ) ; ft - ir 3363.82 cm ( (nh ) ) , 1677.31 cm ( (c = o ) pyrone ) , 1643.58 cm ( amide i band ) , 1621.41 cm ( (c = c ) pyrone ) , 1584.93 cm ( (c = c ) pyrone ) , 1536.21 cm ( amide ii band ) , and mp ( recrystallized from methanol ) 118c . ( 8) preparation of compound 8 ( n-([1,1:4,1-terphenyl]-4-ylmethyl)-3-(benzyloxy)-6-methyl-4-oxo-4h - pyran-2-carboxamide ) . compound 7 ( 90 mg , 0.21 mmol , 1 eq ) is dissolved in toluene in a round bottom flask equipped with a reflux condenser , magnetic stirring bar , and an argon atmosphere . to this mixture are added aqueous potassium carbonate ( 2 m solution , 10 ml ) , triphenylphosphine ( 5.5 mg , 0.021 mmol , 0.1 eq ) , [ 1,1-biphenyl]-4-ylboronic acid ( 41.5 mg , 0.21 mmol , 1 eq ) , and palladium acetate ( 4.6 mg , 0.02 mmol , 0.1 eq ) after which the mixture is heated to 135c with vigorous stirring . the mixture is allowed to stir for 24 h before being extracted with toluene ( 320 ml ) and dried over sodium sulfate and the solvent is removed under reduced pressure . the crude product is purified via column chromatography ( silica , chloroform/1 - 2% methanol ) : h - nmr ( cdcl3 ) 2.37 ( s , 3h ) , 4.444.5 ( d , 2h , j = 5.7 hz ) , 5.34 ( s , 2h ) , 6.29 ( s , 1h ) , 7.107.75 ( m , 18h ) , and 8.12 ( b , 1h ) . compound 8 ( 186.5 mg ) is dissolved in dichloromethane / methanol ( 70/30 , 35 ml ) in a thick - walled glass vessel . after flushing with argon , the vessel is placed in a shaker - type hydrogenation apparatus and placed under 40 psi hydrogen gas for 20 h after flushing with hydrogen gas to remove traces of oxygen . the catalyst is filtered off and the filtrate washed with 10 ml dichloromethane / methanol ( 50/50 ) . the combined organic phase is evaporated in vacuo to yield an orange solid ( 138.5 mg , 90% ) : h - nmr ( cdcl3 ) 2.24 ( s , 3h ) , 4.6 ( s , 2h ) , 6.16 ( s , 1h ) , and 77.8 ( m , 13h ) ; ft - ir 2339.5 cm ( oh ) , 1633.1 cm ( amide i ) , 1591.8 cm ( c = o pyrone ) , 1537.5 cm ( amide ii ) , 1483.9 cm ( c = c pyrone ) , and mp ( recrystallized from toluene ) 253270c . the specificity of the synthesized inhibitor was determined in vitro against a panel of ten human recombinant mmp catalytic domains using a fluorescent assay kit ( bml - ak016 , enzo life sciences ) . briefly , the compound was dissolved in dimethyl sulfoxide ( dmso ) and further diluted in assay buffer ( 50 mm hepes , 10 mm cacl2 , 0.05% brij-35 , and ph 7.5 ) . ten mmps ( mmp-1 , mmp-2 , mmp-3 , mmp-7 , mmp-8 , mmp-9 , mmp-10 , mmp-12 , mmp-13 , and mmp-14 ) were individually incubated with varying concentrations of inhibitor for one hour at 37c , followed by addition of a quenched fluorogenic substrate ( omnimmp fluorogenic substrate mca - pro - leu - gly - leu - dpa - ala - arg - nh2 [ mca is ( 7-methoxycoumarin-4-yl)-acetyl ; dpa is n-3-(2,4-dinitrophenyl)-l---diaminopropionyl ] , 4 m in assay ) . the assays were performed in black , clear bottom , nbs greiner 96-well plates ( 655906 , greiner bio - one ) and fluorescence ( ex = 325 nm , em = 405 nm ) was measured at 60 sec intervals for 30 min with a flexstation ii microplate reader running softmax pro 5.3 ( molecular devices ) . after each fluorescent measurement the well plates were shaken for 2 sec to agitate the reagents . in each experiment we included a blank , a substrate control ( substrate only in assay buffer ) , a positive control ( nngh , 1.3 m final concentration ) , and negative control ( only substrate and inhibitor in assay buffer ) . the latter was implemented to exclude possible interference of the inhibitor with the substrate . to confirm the potency against mmp-3 and mmp-12 , separate assays were performed using human recombinant mmp-3 ( 72006 , anaspec ) and mmp-12 catalytic domains ( 55525 - 1 , anaspec ) and quenched fluorescent substrate qxl 520-pro - leu - ala - tyr - trp - ala - arg - lys(5- fam)-nh2 ( ex = 485 nm , em = 538 nm , 60577 - 01 , anaspec ) . the ic50 values were determined in a single experimental run in duplicate . data analysis and curve fitting for determination of ic50 values were performed in graphpad prism 6 . female c57bl/6j ( 810 weeks old ) mice were purchased from janvier labs ( france ) and mmp-12 knockout mice ( mmp-12 ) ( in c57bl/6j background together with littermate mmp-12 controls ) were obtained from dr . c57bl/6 and mmp-12 mice were housed with 46 mice / cage with ad libitum access to food and water and with a 14-hour light/10-hour dark cycle in a specific pathogen - free animal facility and conventional facility , respectively . all experiments were approved by the ethics committee of the faculty of sciences of ghent university . injection of lipopolysaccharide ( lps ) from salmonella enterica serotype abortus equi ( sigma ) dissolved in pbs . the dose was 200 g/20 g body weight ( the ld100 dose for c57bl/6 mice ) . 100 g mmp-12i ( 1 g/l ) or vehicle control was administered intraperitoneally at different time points : 15 min before and 3 and 6 hours after lps administration . for isolation of total rna from in vivo choroid plexus samples , mice were anesthetized with ketamine / xylazine and perfused with pbs supplemented with bromophenol blue . brains were dissected from the skull and choroid plexus from the third and fourth ventricles were dissected under a dissection microscope . total rna was isolated at different time points after lps injection with the rneasy kit ( qiagen ) . rna concentration and purity were determined spectrophotometrically using the nanodrop nd-1000 ( nanodrop technologies ) . cdna was made by using iscript cdna synthesis kit ( bio - rad ) with 500 ng starting material and qpcr was done using the sensifast sybr no - rox kit ( bioline ) on the light cycler 480 system ( roche ) . expression levels of mmp-12 were normalized to the expression of the two most stable housekeeping genes , ubc and gapdh , which were determined using the genorm housekeeping gene ( hkg ) selection software . one hour before csf isolation , mice were injected i.v . with 75 mg / kg body weight of fitc - labeled dextran ( 4 kda , sigma ) . csf was obtained using the cisterna magna puncture method as described previously . in brief , borosilicate glass capillary tubes ( b100 - 75 - 15 , sutter instruments ) were used to pull needles on the sutter p-87 flaming micropipette puller ( pressure 330 pa , heat index 300 ) . before sampling csf , mice were sedated with ketamine / xylazine . the incision site was sterilized with 70% ethanol , and cisterna magna was exposed by cutting skin and muscle tissue on the posterior side of the skull . the head of the mouse was placed at an angle of 135 degrees and csf was collected by inserting the trimmed needle into the fourth ventricle by piercing the cisterna magna . a csf sample of 2 l was diluted 25-fold in sterile pbs and leakage into this brain compartment was determined by measurement of fluorescence with ex/em = 488/520 nm . culturing primary mouse choroid plexus epithelial cells was done as described . in brief , 29-day - old pups were decapitated and brains were isolated . the cells were dissociated enzymatically by incubating them for 57 min with pronase ( isolated from streptomyces griseus , sigma ) . digestion was stopped by adding an excess of hbss buffer and the cells were washed twice with hbss . the cell pellet was resuspended in dmem - f12 culture medium and plated on laminin - coated plates . two days later , they were shifted to dmem - f12 containing cytosine arabinoside ( ara - c ) to eliminate growth of fibroblasts . purity of the cultures was confirmed by checking the levels of transthyretin ( ttr ) both by qpcr and by immunostaining . primary cpe cells retained the epithelial and the barrier properties which were confirmed by staining for e - cadherin , zo-1 , and occludin . the electric cell - substrate impedance ( ecis ) technique ( applied biophysics ) was used to monitor barrier properties as described before . 250.000 cells per well were seeded onto laminin - coated 8w10e array plates ( applied biophysics ) and the array holder was placed in a standard cell culture incubator ( 37c , 100% humidity , and 5% co2 ) . after ~16 hours , a stable impedance at low and high frequency was obtained and cells were incubated with vehicle or 1 m mmp-12i , followed 1 hour later by vehicle or lps ( 1000 ng / ml ) . compound 9 ( figure 1(a ) ) was assessed for its inhibitory properties towards an array of ten different mmps using a fluorescent in vitro assay . for all tested mmps , the compound is poorly potent against mmp-1 , mmp-3 , mmp-7 , mmp-9 , mmp-10 , and mmp-13 , reflecting ic50 values higher than 50 m ( figure 1(b ) ) . the compound displays slightly better inhibitory activity towards mmp-2 , mmp-8 , and mmp-14 as the apparent ic50 values are situated between 2 m and 50 m . the initial screen revealed an apparent ic50 value for mmp-12 below 2 m and when performing a detailed dose - response experiment using a more specific substrate for mmp-12 ( substrate xiii , anaspec ) a nonlinear regression analysis indicated that at higher inhibitor concentrations an inhibitory plateau ( 72 1.75% ) is reached with a relative ic50 of 177 nm ( figure 1(c ) ) . although the use of a different substrate can potentially influence the kinetic readout , a comparison between the fluorescent in vitro assay kit and the use of substrate xiii resulted in similar inhibitor potencies ( data not shown ) , which justifies the use of the more specific substrate for mmp-12 . taken together , these data indicate that the synthesized compound is a potent and fairly specific inhibitor of mmp-12 and therefore from section 3.2 onwards it will be referred to as mmp-12i . the discrepancy between puerta et al . and this study regarding the inhibitory selectivity towards mmp-12 over mmp-3 could in part be explained by the strong ph - dependence of inhibitor potency for human mmp-3 . both mmp-3 and mmp-12 have a deep pocket s1 subsite in which the terphenyl can reside . however , mmp-3 is uniquely characterized by a sharp optimum at ph 6 for efficient catalysis and inhibition and this is drastically reduced at neutral ph by structural changes in the s1 pocket and ionization of inhibitor residues . in contrast to the study of puerta and colleagues , the compound was tested in physiologically relevant ph 7.5 to accommodate a large array of mmps . importantly , the ph - dependence of inhibitor - mmp-3 interactions typically gives rise to a change in ic50 values not larger than one order of magnitude as opposed to the three orders of magnitude we established here , so other factors might be at play . because of this and the observed potency towards mmp-12 we further validated the biological relevance of the compound in the context of mmp-12 . endotoxemia , that is , systemic administration of lipopolysaccharide ( lps ) , induces systemic inflammation and eventually lethality in mice . we have previously shown that mice can be protected from lps - induced lethality by administration of a broad spectrum mmp inhibitor bb-94 and several mmps appeared to play a role in the lps - induced lethality [ 8 , 1113 ] . here , we studied the effect of mmp-12 deficiency on the lps sensitivity by using mmp-12 mice . as shown in figure 2(a ) , mmp-12 mice display a partial protection to an ld100 dose of lps , indicating that mmp-12 plays a detrimental role in the lps - induced lethality . to verify this , we treated wild type mice with the mmp-12i and challenged them with lps ( figure 2(b ) ) . mmp-12i treatment resulted in a significant reduction in lps - induced lethality compared with vehicle treated mice , which again demonstrates that the compound most likely targets mmp-12 . systemic inflammation is known to be associated with loss of blood - brain barrier integrity and this has been correlated with lethality . next to the most studied endothelial blood - brain barrier ( bbb ) , we have previously shown that the blood - cerebrospinal fluid barrier ( bcsfb ) which is formed by the choroid plexus epithelium ( cpe ) is also affected in response to systemic lps administration . mrna expression analysis of choroid plexus tissue revealed 500-fold upregulation of mmp-12 upon systemic lps administration ( figure 2(c ) ) , associated with an increase in bcsfb permeability ( figure 2(d ) ) . interestingly , the loss of bcsfb permeability was significantly reduced when endotoxic mice were treated with our mmp-12i ( figure 2(d ) ) . therefore , primary cpe cells were isolated and plated onto electrical cell impedance sensing ( ecis ) arrays containing gold electrodes . ecis is a real - time , label - free , impedance - based method used to study the barrier properties of cells grown in culture . after reaching confluency , cells were incubated with lps or lps supplemented with mmp-12i and compared to untreated cells . lps resulted in a drop in normalized resistance compared to control and this was significantly reduced in the presence of mmp-12i ( figure 2(e ) ) . these data clearly show that mmp-12 plays a role in the observed lps - induced loss of bcsfb integrity . in summary , we report the synthesis and biological evaluation of a hydroxypyrone - based mmpi with excellent potency and specificity towards mmp-12 . both in vivo and in vitro studies on the effects of the target compound on endotoxemia and bcsfb integrity clearly indicate a role for mmp-12 in these inflammatory processes and establish the use of this compound in a biological context .

a hydroxypyrone - based matrix metalloproteinase ( mmp ) inhibitor was synthesized and assayed for its inhibitory capacity towards a panel of ten different mmps . the compound exhibited selective inhibition towards mmp-12 . the effects of inhibition of mmp-12 on endotoxemia and inflammation - induced blood - cerebrospinal fluid barrier ( bcsfb ) disruption were assessed both in vitro and in vivo . similar to mmp-12 deficient mice , inhibitor - treated mice displayed significantly lower lipopolysaccharide- ( lps- ) induced lethality compared to vehicle treated controls . following lps injection mmp-12 mrna expression was massively upregulated in choroid plexus tissue and a concomitant increase in bcsfb permeability was observed , which was restricted in inhibitor - treated mice . moreover , an lps - induced decrease in tight junction permeability of primary choroid plexus epithelial cells was attenuated by inhibitor application in vitro . taken together , this hydroxypyrone - based inhibitor is selective towards mmp-12 and displays anti - inflammatory activity in vitro and in vivo .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusion

PMC4624118

obesity as a common problem in menopausal women is a major risk factor for the development of cardiovascular diseases . this is due to the importance of estrogen in the determination of body fat distribution . previous studies showed that deficiency of this hormone leads to an accumulation of visceral fat , and consequently , a decrease in insulin sensitivity . obesity is often associated with cardiovascular disease risk factors such as hypertension , dyslipoproteinemia , diabetes , and elevated inflammation markers [ 79 ] . accumulating evidence suggests that altered vitamin d homeostasis may also contribute to an increased cardiovascular disease risk in obese subjects . there are positive correlations between low 25-hydroxyvitamin d [ 25(oh)d ; < 43 nmol / l ] and obesity as well as between 25(oh)d < 3337.5 nmol / l and cardiovascular mortality . vitamin d has a protective effect on bones , anticancer and anti inflammatory effects and its receptors are present in pancreatic b cells and insulin sensitive tissues including skeletal muscle tissues . vitamin d repletion improves insulin and glucose homeostasis in animal models of vitamin d deficiency . it has also been reported that vitamin d deficiency in the blood is greatly associated with various metabolic factors related to diabetes such as hyperglycemia and insulin resistance , but data supporting this relationship are sparse . there are some studies reporting physical activity as an important environmental factor associated with body weight regulation and increasing physical activity has become an important aspect of non - pharmacological strategies to control obesity , weight gain and blood glucose in addition to preventing progression from impaired glucose tolerance . due to this connection , the american diabetes association ( ada ) recommends aerobic exercise of medium intensity such as 150 minutes of walking per week , or 75 minutes of high intensity aerobic exercise per week for patients with type 2 diabetes . previous studies showed that only 28% of postmenopausal women perform regular physical activity . despite the beneficial effects of physical activity , there are many complications to achieving the predicted improvement in health . first of all , menopausal women are predisposed to osteoporosis and tend to avoid involvement in different kinds of physical activity . second , it was recently reported that exercise is not that effective in some obese people . the mechanisms by which the central nervous system or hormones interact with metabolic pathways and restrict weight loss are not yet understood . here , we assumed that vitamin d could be one of the determining factors in achieving the health benefits of exercise . therefore , the authors of this paper intended to investigate the effects of implementing vitamin d intake for 8 weeks in addition to aerobic exercise training on the plasma lipid profiles and insulin resistance in ovariectomized rats . forthy female wistar rats weighing 240255 g were housed four per cage and fed standard - pellet rat chow and tap water ad libitum . the 12-h:12-h light cycle ( in which all fluorescent lighting was shielded , preventing the endogenous production of vitamin d3 ) began at 07.00 and the room temperature was maintained at 2124c . the experiments described in this study were conducted according to the policy of the iranian convention for the protection of vertebrate animals used for experimental and other scientific purposes , and the protocol was approved by the ethics committee of guilan university of medical sciences , rasht , iran ( irb number ; 88038 ) . body weight , food intake and visceral fat were measured using a scale ( sartorius , germany ) accurate to 0.1 g. all rats were weighed once a week , between 09.00 and 11.30 . also , food intake was monitored weekly . once a week , the food was weighed before it was given to the animals and again at the same time the next day , after it was removed from the cages . to measure the food intake , an equal amount of food ( 20 g / day / rat ) was given to each rat in each cage in each identical group , and food consumption was measured by subtracting the weight of the remaining uneaten food . the differences between the weights were divided by the number of rats in the cage . after 8 weeks of exercise , rats were anesthetised for the measurement of body length ( nose - to - anus ) . two month dietary manipulation of vitamin d was carried out using cholecalciferol ( vit d3 ) added to an isocaloric diet in the following amounts ( based on pilot studies intended to mimic the range of human levels ) : aerobic training with vitamin d supplementation = 10000 iu / kg ( 9000 iu by injection and 1000 iu by food intake ) per week , vitamin d supplementation = 10000 iu / kg ( 9000 iu by injection and 1000 iu by food intake ) per week , control vit d = 1000 iu / kg food a week , aerobic training vit d = 1000 iu / kg food per week . rats in the at group were trained to run on a motor - driven rodent treadmill ( iranian model , seven lanes , designed by sports sciences research center , tehran , iran ) from the 3rd week of experiment ( 16 weeks old ) . during the first week of training , rats began to practice walking at the speed of 6 m / min , 10 min / day , with no inclination of the treadmill . exercise training consisted of running on the treadmill for 3 days / week for 8 weeks . rats progressed from running for 15 min / day at 15 m / min , 0% slope , up to 40 min / day at 25 m / min , 10% slope for the last 4 weeks . two weeks after acclimatization , 16-week - old rats were separated into ovx ( n = 32 ) and sham - operated control ( sham ; n = 8) groups . ovx rats were subdivided into four groups : aerobic training ( ovx + at ; n = 8) , vitamin d supplementation ( ovx + vit d ; n = 8) , aerobic training with vitamin d supplementation ( ovx + at + vit d ; n = 8) and control ( ovx + c ; n = 8) group . rats were ovariectomized and sham - operated under general anesthesia with an intraperitoneal injection of ketamine ( 50 mg / ml ; rotex medica , germany ) and xylazine ( 20 mg / ml ; alfasan , the netherlands ) at a ratio of 4:1 , according to the technique described by shinoda and colleagues . all trained rats were restrained from training and food was removed from the animals cages at least 12 h before sacrifice . after complete anesthesia , the abdominal cavity was rapidly opened and blood samples were drawn from the inferior vena cava . the serum was separated by centrifugation ( 4000 rpm for 15 min ) and stored at 80c for later biochemical and hormonal measurements . the concentrations of triglyceride , total cholesterol , hdl - cholesterol and ldl - cholesterol in the plasma were measured using enzymatic analysis kits ( asan pharmaceuticals , hwasung , korea ) . the serum glucose concentration was determined with an enzymatic ( god - pap , glucose oxidaseamino antipyrine ) colorimetric assay ( pars azmoun , tehran , iran ) and the serum insulin concentration was measured with a rat insulin elisa kit ( drg , usa ) . homa - ir ( fasting insulin ( m/ml)fasting glucose ( mmol / l)/22.5 ) was used to estimate the insulin resistance . all data are presented as mean standard error . before statistical analysis , the normal distribution and homogeneity of the variances comparisons within groups ( after 8 weeks ) were performed by repeated measure anova followed by tukey s post - hoc test . the sas software version 9.1 was used for data analysis , with the level of significance set at 5% ( p < 0.05 ) . forthy female wistar rats weighing 240255 g were housed four per cage and fed standard - pellet rat chow and tap water ad libitum . the 12-h:12-h light cycle ( in which all fluorescent lighting was shielded , preventing the endogenous production of vitamin d3 ) began at 07.00 and the room temperature was maintained at 2124c . the experiments described in this study were conducted according to the policy of the iranian convention for the protection of vertebrate animals used for experimental and other scientific purposes , and the protocol was approved by the ethics committee of guilan university of medical sciences , rasht , iran ( irb number ; 88038 ) . body weight , food intake and visceral fat were measured using a scale ( sartorius , germany ) accurate to 0.1 g. all rats were weighed once a week , between 09.00 and 11.30 . also , food intake was monitored weekly . once a week , the food was weighed before it was given to the animals and again at the same time the next day , after it was removed from the cages . to measure the food intake , an equal amount of food ( 20 g / day / rat ) was given to each rat in each cage in each identical group , and food consumption was measured by subtracting the weight of the remaining uneaten food . the differences between the weights were divided by the number of rats in the cage . after 8 weeks of exercise , rats were anesthetised for the measurement of body length ( nose - to - anus ) . two month dietary manipulation of vitamin d was carried out using cholecalciferol ( vit d3 ) added to an isocaloric diet in the following amounts ( based on pilot studies intended to mimic the range of human levels ) : aerobic training with vitamin d supplementation = 10000 iu / kg ( 9000 iu by injection and 1000 iu by food intake ) per week , vitamin d supplementation = 10000 iu / kg ( 9000 iu by injection and 1000 iu by food intake ) per week , control vit d = 1000 iu / kg food a week , aerobic training vit d = 1000 iu / kg food per week . rats in the at group were trained to run on a motor - driven rodent treadmill ( iranian model , seven lanes , designed by sports sciences research center , tehran , iran ) from the 3rd week of experiment ( 16 weeks old ) . during the first week of training , rats began to practice walking at the speed of 6 m / min , 10 min / day , with no inclination of the treadmill . exercise training consisted of running on the treadmill for 3 days / week for 8 weeks . rats progressed from running for 15 min / day at 15 m / min , 0% slope , up to 40 min / day at 25 m / min , 10% slope for the last 4 weeks . two weeks after acclimatization , 16-week - old rats were separated into ovx ( n = 32 ) and sham - operated control ( sham ; n = 8) groups . ovx rats were subdivided into four groups : aerobic training ( ovx + at ; n = 8) , vitamin d supplementation ( ovx + vit d ; n = 8) , aerobic training with vitamin d supplementation ( ovx + at + vit d ; n = 8) and control ( ovx + c ; n = 8) group . rats were ovariectomized and sham - operated under general anesthesia with an intraperitoneal injection of ketamine ( 50 mg / ml ; rotex medica , germany ) and xylazine ( 20 mg / ml ; alfasan , the netherlands ) at a ratio of 4:1 , according to the technique described by shinoda and colleagues . all trained rats were restrained from training and food was removed from the animals cages at least 12 h before sacrifice . after complete anesthesia , the abdominal cavity was rapidly opened and blood samples were drawn from the inferior vena cava . the serum was separated by centrifugation ( 4000 rpm for 15 min ) and stored at 80c for later biochemical and hormonal measurements . the concentrations of triglyceride , total cholesterol , hdl - cholesterol and ldl - cholesterol in the plasma were measured using enzymatic analysis kits ( asan pharmaceuticals , hwasung , korea ) . the serum glucose concentration was determined with an enzymatic ( god - pap , glucose oxidaseamino antipyrine ) colorimetric assay ( pars azmoun , tehran , iran ) and the serum insulin concentration was measured with a rat insulin elisa kit ( drg , usa ) . homa - ir ( fasting insulin ( m/ml)fasting glucose ( mmol / l)/22.5 ) was used to estimate the insulin resistance . all data are presented as mean standard error . before statistical analysis , the normal distribution and homogeneity of the variances comparisons within groups ( after 8 weeks ) were performed by repeated measure anova followed by tukey s post - hoc test . the sas software version 9.1 was used for data analysis , with the level of significance set at 5% ( p < 0.05 ) . the results for body weight and food intake changes between and within groups are shown in figs . 1 and 2 respectively . initial body weights and food intakes in the ovx groups were not significantly different in this study . compared with the sham rats , the ovx rats were found to have higher body weight intakes ( p 0.05 ) . three weeks after the beginning of the study , there was significant difference in body weight between the groups ( fig . 1 ) . the repeated measures anova test showed a significant reduction in body weight in ovx + at + vit d , ovx + at and ovx + vit d groups during the experiment , while the body weights of the ovx - c and sham groups significantly increased after 8 weeks compared with the initial body weights . also , food intake significantly increased in the ovx + c and sham groups over time , but significantly decreased in the ovx + at + vit d group ( fig . interestingly , the mean body weight and food intake from the third week in the control group was significantly higher than in the other groups ( fig . 1 and 2 ) . the tc , tg , hdl - c and ldl - c levels are shown in table 1 . the tc levels were significantly higher in the c group than in the other groups ( 11.74% , p = 0.002 ) , while the tc levels were significantly lower in the ovx + at + vit d group than in the other groups ( 11.99% , p = 0.001 ) . the ovx + at + vit d group displayed significantly lower tg levels than the other groups ( 11.30% , p = 0.001 ) . similarly , the ovx + at , ovx + vit d and sham groups had significantly lower tg levels than the c group ( 15.01% , p = 0.001 ; 1.51% , p = 0.003 ; 4.34% , p = 0.002 ) . the hdl - c levels were significantly higher in the ovx + at + vit d and ovx + at groups than in the other groups ( 22.40% , p = 0.001 and 12.06% , p = 0.043 ) ( table 1 ) . furthermore , the hdl - c levels were significantly lower in the c group compared with the other groups ( 17.48% , p = 0.002 ) . the ldl - c levels were significantly lower in the ovx + at + vit d group than in the other groups ( 19.74% , p = 0.001 ) and were significantly much higher in the c group compared with the other groups ( 25.74% , p = 0.001 ) . the visceral fat level was significantly lower in the ovx + at + vit d , ovx + vit d , ovx + at and sham groups compared with the control group ( 13.10% , p = 0.001 ; 2.83% , p = 0.003 ; 10.48% , p = 0.004 and 10.69% , p = 0.003 , respectively ) . glucose , serum insulin and homa - ir levels decreased significantly by 5.82% , 15.56% and 20.94% , respectively , in ovx + at + vit d compared with the other groups ( p = 0.001 ; table 1 ) . the purpose of this study was to examine the interaction between aerobic exercise training and vitamin d supplementation and its effect on plasma lipid profiles and insulin resistance in ovariectomized rats . in the study , ovariectomy resulted in a significant increase in body weight and also an increase in visceral fat , confirming previous findings , this indicates that the lack of estrogen following ovariectomy influences body weight , partially by increasing visceral fat . estrogen plays a crucial role in distributing visceral fat , probably by changing lipoprotein lipase activity . our study revealed that exercise training decreases body weight , which has also been reported in many studies . on the other hand , there is evidence showing lack of weight reduction after exercise , especially in menopausal women . it has been reported that the concentration of serum vitamin d has an effect on muscular activity , the number and size of muscular fibers and also the fat free mass . the present study showed that treatment with ovx - at & vit d for 8 weeks leads to more weight loss ( 21% ) compared with exercise alone ( 7% ) . additionally , mean food intake from week three in the control group was significantly higher than other groups . reported that in a study comparing exercise training and vitamin d intake ( 400 iu / day ) over 9 months in elderly women , the fat free mass and the muscle strength in the exercise training group significantly increased , but there were no significant changes in fat free mass and muscle strength in the vitamin d intake group . it has been reported that unlike vitamin d intake , regular aerobic training effects positive changes in body weight . exercise training targeting patients with diabetes improve their health and has positive effects on body weight . on the other hand , there have also been research results indicating that significant changes in body weight were not apparent despite participation in exercise training . according to body weight and food intake results obtained in this study , the most significant reduction was in the group with aerobic training combined with vitamin d. the mechanism by which an increase in the concentration of vitamin d in the body affects body composition is difficult to explain . however , some studies reported no effect from vitamin d on body weight change and energy expenditure and little improvement in cardiovascular risks with cholecalciferol . interestingly , vitamin d is fat - soluble and is readily stored in adipose tissue ; it is sequestered after absorption and stored in tissues such as fat and muscle . this fate of vitamin d has been demonstrated by injecting radio - labeled vitamin d3 into individuals and monitoring the highest levels of biological activity and radioactivity in the fat tissue . 1,25-dihydroxyvitamin d , inversely , modulates adipokine expression , inhibits anti - inflammatory cytokine expression , reduces monocyte recruitment by human pre - adipocytes , and restores glucose uptake in adipocytes . 1,25-dihydroxyvitamin d has a role in human adipose tissue because it is the active form of the vitamin d3 metabolite . vitamin d receptors ( vdr ) present in adipocytes allow the suppression of parathyroid hormone ( pth ) levels . the pth excess observed in menopausal women with both primary and secondary hyperparathyroidism may promote weight gain by impeding catecholamine - induced lipolysis . a previous study showed that vit d has a positive effect on muscle mass , especially on fat free mass , and also on muscle activity . we assumed that an increase in muscle mass could elevate metabolism and decrease fat mass . regarding the one - way anova test , we showed that the tg , tc , and ldl - c levels were significantly decreased with increased aerobic training and vitamin d intake . also , the hdl - c levels were significantly higher in the ovx + at + vit d and ovx + at groups compared with the other groups . several studies have revealed the importance of nutritional education aimed at preventing cardiovascular disease ( cvd ) in postmenopausal women . the present study confirmed this with the observation of a significant decline in body weight , food intake , tg , tc , and ldl - c levels as well as increases in the hdl - c level by aerobic training and vit d. interestingly , weight loss parallels the reduction of cardiovascular risk factors ( tg , tc , ldl - c ) and hdl - c levels were improved in the exercise with vitamin d supplemented group compared with the exercise only group . few studies have investigated the relationship between serum levels of vitamin d and lipid profiles . major et al . reported that in obese middle - aged women taking 200 iu of vitamin d per day for 15 weeks , tc , ldl - c , and hdl - c concentrations were significantly reduced . rejnmark and colleagues performed a study among 82 healthy postmenopausal women who had been treated with either 40 mg / day simvastatin or a placebo for 1 year , in which vitamin d , tg , and ldl levels were measured at baseline and after 26 weeks of treatment . in this study , simvastatin showed no effect on vitamin d status , but reduced the serum levels of tg and ldl . these results suggest that the serum concentration of tg is inversely associated with the serum level of 25(oh ) d . in contrast , kota and colleagues showed no relationship between serum levels of 25(oh ) d and tg or hdl cholesterol in healthy subjects . it was suggested that vitamin d has both direct and indirect effects on modifying the lipid profile and that the effect of vitamin d on decreasing serum levels of tg , tc and ldl may occur through regulatory action that increases the activity of lipoprotein lipase in adiposity . in our study , exercise led to improvements in lipids profile as well . in agreement with our results , greene et al . also reported that obese middle - aged men and women participating in endurance exercise at 70% of vo2max , 3 times per week for 12 weeks had significant improvements in lipid profiles . sung & bae reported a similar finding in diabetic elderly men participating in walking exercise at 6575% of hrmax for 50 minutes a day , 3 times a week for 24 weeks . taken together , aerobic training is considered effective for inducing positive changes in blood lipid . several mechanisms are suggested to explain the effect of vitamin d on lipids , including its reducing role in fatty acid absorption via the formation of insoluble calcium - fatty complexes in the gut . by decreased absorption of fat , particularly saturated fatty acids , it is expected that serum levels of total and ldl cholesterol will be reduced . in addition , vitamin d can increase the conversion of cholesterol to bile acids due to its ability to bind with bile acids . however , the effect of enteric vitamin d and calcium on lipid absorption is very limited and it does not have a significant effect on lipid profiles . on the other hand , in vitro studies have demonstrated that pth reduces lipolysis ( through increasing the level of cytosolic calcium ) and increases the expression of fatty acid synthesis . based on existing studies , it is known that the accumulation of visceral fat in postmenopausal women is the main cause of insulin resistance and metabolic syndrome . it has been reported that the accumulation of visceral fat increases the secretion of adipocytokine and has a negative effect on hypertension , diabetes and cardiovascular diseases . previous studies reported that vitamin d deficiency often appeared in cases of obesity and the concentration of 25(oh)d shows a negative correlation with visceral fat . in our study , visceral fat showed more reduction in ovx + at + d compared with other groups . interestingly , the reduction of visceral fat in the group receiving co - treatment of exercise and vit d was much higher than with exercise alone . in this regard , bacchi et al . reported that for middle - aged men and women with diabetes conducting aerobic exercise using a tread mill or cycle at 6065% of hrr , 60 minutes per day , 3 times per week for 16 weeks , the abdominal visceral fat area and subcutaneous fat area were significantly reduced . while inconsistent , wamberg et al . reported that women taking vitamin d ( 7,000 iu per day over 26 weeks ) showed no significant changes in subcutaneous fat mass and visceral fat mass generally , the research results show that aerobic exercise training and adequate intake of vitamin d positively affect the body weight , visceral fat and plasma lipid profiles during menopause and also reduce metabolic syndrome risk factors . in addition , the results of our study showed a significant improvement in insulin resistance in the ovx + d + at group ( fig 2 , b and , d ) . reported that people who have vit d insufficiency ( < 30 ngml ) and people with deficiency ( < 20 ngml ) < incomplete sentence > . also , vonhurst et al . reported that as a result of taking 4,000 iu of vitamin d every day for 26 weeks , the insulin and blood glucose levels of patients with diabetes were significantly reduced . although our previous studies and others showed adequate effectiveness by regular physical activity in improving insulin resistance , ovx + at + d showed a better response . there are some mechanisms for the effects of vitamin d : the presence of vitamin d receptors on pancreatic cells , vitamin d - activating 1hydroxylase is expressed in pancreatic cells , the vitamin d response element in the insulin gene due to the presence of vitamin d receptors in skeletal muscle and the fact that 1,25(oh)d increases the transcription of insulin receptor genes and also suppresses the renin gene , reducing hyperglycemic - induced increases in renin levels in pancreatic cells . blockade of renin - angiotensin activity has been proposed as a novel target for the treatment of diabetes . the protective effects of vitamin d on obesity may be due to well known effects of vitamin d such as its anti - inflammatory properties , its effects on calcium and phosphorus metabolism and its regulation of the insulin receptor gene . it seems that vitamin d increases the calcium content of cells , in turn leading to the increased transport of glucose into the muscle . vitamin d also regulates nuclear ppar ( peroxisome proliferative activated receptor ) , which has an important role in insulin sensitivity . it would be useful though to undertake further studies to discover more about the mechanism and the effect of vitamin d on both alpha and islet beta - cell functions and also on the mechanisms that determine insulin resistance . aerobic training and vitamin d supplementation contribute to increases in the metabolic rate by improving cardiovascular capacity and suppressing pro - inflammatory cytokine production , in addition to increasing the release of nitric oxide and the consumption of free fatty acids , thus enhancing insulin sensitivity . in general , inadequate vitamin d may promote greater adiposity through other metabolic effects , such as the regulation of pth and the modulation of adipogenesis . moderate to severe vitamin d deficiency leads to increased pth , which may promote an increase in free intracellular calcium in adipocytes and thereby enhance lipogenesis . the question that needs to be answered is whether or not vitamin d deficiency prevents the beneficial effects of exercise especially after menopause . recently , numerous interventional and epidemiological studies have shown that calcium and vitamin d consumption effectively reduce abdominal fat and metabolic syndrome risk . it has been reported that vitamin d plays an essential role in the homeostasis control of calcium . vitamin d was closely connected to the metabolic mechanism of calcium , and increased blood calcium concentration consequently affects blood lipid concentration and insulin resistance . one limitation of the present study was the lack of a group of ovariectomized animals treated with hormone replacement therapy to confirm the positive effects of at and vit d on plasma lipid profiles and insulin resistance . further studies should be conducted to more accurately elucidate and characterize the metabolic energy imposed by this aerobic training protocol and vitamin d supplementation in rats and humans . in conclusion , aerobic training and vitamin d supplementation successfully attenuate some metabolic syndrome profiles and might prevent cardiovascular risk factors caused by ovariectomy . from the clinical point of view , this study suggests that vit d supplementation is a very useful strategy to prevent obesity and menopause - associated metabolic disturbances .

purposethe purpose of this study was to determine the interaction effects of aerobic exercise training and vitamin d supplementation on indices of obesity and plasma lipid profiles in ovariectomized ( ovx ) rats.methodsforty female wistar rats were divided into 5 groups : aerobic training ( 3 days / week for 8 weeks ; at ; n = 8) , aerobic training and vitamin d supplementation ( ovx + at + vit d ; n = 8) , vitamin d supplementation ( ovx + vit d ; n = 8) , ovariectomized control ( ovx + c , n = 8) and sham ( n = 8) . after blood sampling , visceral fat was taken from the abdominal cavity and weighed immediately . data was statistically analyzed by one - way anova and repeated measure anova tests with a 0.05 significance level.resultsbody weight , visceral fat , bmi and food intake decreased significantly in ovx + at + vit d ( p < 0.001 ) ; whereas these variables increased significantly in ovx + c ( p < 0.001 ) and sham ( p < 0.023 ) groups . at the end of two - months of follow - up , we observed significant differences in tc , tg , hdl - c , ldl - c , glucose , insulin , and homa - ir in all groups.conclusionit seems that aerobic training with vitamin d , due to the involvement of muscle mass and exposure to dynamic pressure on the bones and muscles , increased energy expenditure , stimulated insulin exudation and glucose homeostasis , decreased insulin resistance and improved the lipid profile in ovariectomized rats .

INTRODUCTION METHODS Animal care Body weight and food intake Vitamin D supplementation Protocol for exercise training Groups and surgery Blood analysis Assessment of concentrations of plasma lipids Statistical analysis RESULTS DISCUSSION CONCLUSIONS

PMC3830968

, radical retropubic prostatectomy ( rrp ) has been the most common form of surgical treatment for prostate cancer . however , in the last 8 years , there has been a paradigm shift in surgically treated prostate cancer with the adaptation of robot - assisted laparoscopic prostatectomy ( ralp ) . because of the presence of three - dimensional magnification and tools with 7 degrees of freedom that can duplicate hand movements with high accuracy , ralp has gained notable popularity in both the united states and europe ; furthermore , it is estimated that more than 75% of radical prostatectomies are performed by use of the da vinci robotic platform ( intuitive surgical inc . , sunnyvale , ca , usa ) . the feasibility , safety , and early functional efficacy of ralp have been well documented . a recent population - based analysis comparing ralp and rrp procedures undertaken between 2003 and 2005 concluded that men undergoing ralp experienced significantly fewer 30-day complications , blood transfusions , and anastomotic strictures and shorter lengths of stay . in another study , other studies have also consistently shown that blood loss and length of hospital stay are less in patients who undergo ralp than in those who undergo rrp . however , there is lack of uniformity in documenting and reporting complications that may result in incomplete capture of data and may make comparisons among different surgical approaches or institutional series problematic . menon et al . compared the complications at one institution between rrp performed by one group of surgeons and ralp performed by a different group of surgeons . although the study was prospective and covered the same period , inter - surgeon differences in technique may have weakened the ability to objectively compare differences in complications . moreover , most studies did not use a formal reporting system for complications or a uniform grading system . in this study , we compared the complications of rrp with ralp performed by a single surgeon using the modified clavien system . three hundred forty - one rrps and 524 ralps were performed by a single surgeon ( c.s.k . ) between july 2007 and august 2012 . the conducting surgeon had substantial experience with rrp but no experience with ralp at the start of the study period ; in fact , he did not have any experience with laparoscopic radical prostatectomy . in korea , the cost to the hospital for ralp is much greater than that for rrp because rrp is covered by national insurance , whereas ralp is not . therefore , the choice of surgical method was made on the basis of counseling with patients , considering the severity and extent of their disease and their characteristics . the preoperative workup for both groups consisted of serum prostate - specific antigen measurement , 10- to 12-core biopsies for cancer detection , magnetic resonance imaging , and a bone scan . prostate volume and clinical stage were evaluated by transrectal ultrasonography and digital rectal examination , respectively . comorbidities ( e.g. , diabetes mellitus , hypertension , chronic obstructive pulmonary disease , and cardiovascular disease ) and social history were assessed . tumors were graded histologically according to the gleason grading system , and the pathological stage was defined according to the tnm staging classification . on the fifth to sixth postoperative day , cystography with anteroposterior and lateral views was performed to evaluate the anastomotic tightness . if there was no leak as evidenced by cystography , the urethral catheter was removed . briefly , a lower midline abdominal incision was made , entering the space of retzius . thereafter , for the cases in which a nerve - sparing procedure was attempted , the surgeon dissected the lateral aspects from the prostate allowing the neurovascular bundles to retract laterally . the attachments between the bladder and the prostate were then divided and the surgical specimen was removed . the vesicourethral anastomosis with six separate sutures was typically performed over a 20-fr urethral catheter . in ralp , pneumoperitoneum was established by using a veress needle and five trocars were then inserted . the prostate was dissected with an antegrade approach , beginning with a bladder neck - sparing procedure . for all potent patients , a continuous suture was performed during the creation of the vesicourethral anastomosis with two 3 - 0 monocryl sutures tied together , as described by van velthoven . medical and surgical complications were retrospectively collected for all patients through a combination of institutional electronic medical records , including operative and nursing notes , discharge summaries , outpatient and emergency room visits , and written correspondence with patients regarding complications . any complications that occurred within 90 days of surgery were analyzed and categorized as either medical or surgical . this system determines the severity of a complication by using a scale with five grades . grade i complications are designated as any deviation from the normal postoperative course that do not require extra therapy ( with the exception of antiemetic , antipyretic , analgesic , and antidiarrheal drugs ) . grade ii complications necessitate pharmacologic treatment with drugs other than the drugs mentioned for the grade i complications . grade iii complications are defined as complications necessitating surgical , endoscopic , or radiologic intervention . this grade is subdivided into grades iiia and iiib on the basis of the need for general anesthesia . grade iv complications are life - threatening and necessitate intensive care , often leaving the patient with residual disability . a grade v complication represents the death of a patient as the result of the complication . the two - tailed pearson chi - square test was used to assess differences in the variables between the rrp and ralp groups . three hundred forty - one rrps and 524 ralps were performed by a single surgeon ( c.s.k . ) between july 2007 and august 2012 . the conducting surgeon had substantial experience with rrp but no experience with ralp at the start of the study period ; in fact , he did not have any experience with laparoscopic radical prostatectomy . in korea , the cost to the hospital for ralp is much greater than that for rrp because rrp is covered by national insurance , whereas ralp is not . therefore , the choice of surgical method was made on the basis of counseling with patients , considering the severity and extent of their disease and their characteristics . the preoperative workup for both groups consisted of serum prostate - specific antigen measurement , 10- to 12-core biopsies for cancer detection , magnetic resonance imaging , and a bone scan . prostate volume and clinical stage were evaluated by transrectal ultrasonography and digital rectal examination , respectively . comorbidities ( e.g. , diabetes mellitus , hypertension , chronic obstructive pulmonary disease , and cardiovascular disease ) and social history were assessed . tumors were graded histologically according to the gleason grading system , and the pathological stage was defined according to the tnm staging classification . on the fifth to sixth postoperative day , cystography with anteroposterior and lateral views was performed to evaluate the anastomotic tightness . if there was no leak as evidenced by cystography , the urethral catheter was removed . briefly , a lower midline abdominal incision was made , entering the space of retzius . thereafter , for the cases in which a nerve - sparing procedure was attempted , the surgeon dissected the lateral aspects from the prostate allowing the neurovascular bundles to retract laterally . the attachments between the bladder and the prostate were then divided and the surgical specimen was removed . the vesicourethral anastomosis with six separate sutures was typically performed over a 20-fr urethral catheter . in ralp , pneumoperitoneum was established by using a veress needle and five trocars were then inserted . the prostate was dissected with an antegrade approach , beginning with a bladder neck - sparing procedure . for all potent patients , a continuous suture was performed during the creation of the vesicourethral anastomosis with two 3 - 0 monocryl sutures tied together , as described by van velthoven . medical and surgical complications were retrospectively collected for all patients through a combination of institutional electronic medical records , including operative and nursing notes , discharge summaries , outpatient and emergency room visits , and written correspondence with patients regarding complications . any complications that occurred within 90 days of surgery were analyzed and categorized as either medical or surgical . this system determines the severity of a complication by using a scale with five grades . grade i complications are designated as any deviation from the normal postoperative course that do not require extra therapy ( with the exception of antiemetic , antipyretic , analgesic , and antidiarrheal drugs ) . grade ii complications necessitate pharmacologic treatment with drugs other than the drugs mentioned for the grade i complications . grade iii complications are defined as complications necessitating surgical , endoscopic , or radiologic intervention . this grade is subdivided into grades iiia and iiib on the basis of the need for general anesthesia . grade iv complications are life - threatening and necessitate intensive care , often leaving the patient with residual disability . a grade v complication represents the death of a patient as the result of the complication . the two - tailed pearson chi - square test was used to assess differences in the variables between the rrp and ralp groups . all statistical analyses were performed by using ibm spss ver . 21.0 ( ibm co. , armonk , ny , usa ) . the clinical stage , d'amico classification , pathological stage , pathological gleason grade , and treatment characteristics of all patients are also shown in table 1 . the mean hospital stay ( 7.9 days vs. 10.1 days , p<0.001 ) and mean urethral catheter duration ( 6.2 days vs. 7.5 days , p<0.001 ) were significantly shorter in the ralp group than in the rrp group , respectively . the postoperative complications for the two groups by use of the clavien classifications are shown in table 2 . the complication rate of the ralp group was lower than that of the rrp group ( 27.3% vs. 68.0% , respectively ) . among them , there were more major complications defined as clavien grade iii or more in the rrp group ( 3.4% vs. 7.6% , respectively ) . in the ralp group , clavien grade iii complications included anastomotic leakage , infected lymphocele , wound dehiscence , surgical reintervention , and anal abscess . clavien grade iii complications in the rrp group also included anastomotic leakage , wound dehiscence , and urethral dilation . concerning clavien grade iv complications , one patient in the rrp group was taken to the intensive care unit ( icu ) after surgery because of the occurrence of stroke . three cases of stroke and icu care owing to small bowel injury and postoperative bleeding were included in the clavien grade iv complications in the ralp group . the incidence of medical complications ( table 3 ) was not significantly different between the two groups . the rates of cardiac problems , pneumonia , urinary tract infections , respiratory distress , respiratory failure , acute renal failure , and cerebrovascular accidents were similar in both groups . urinary retention was more common in the rrp group ( 7.0% vs. 2.7% , respectively ; p=0.002 ) . the need for perioperative blood transfusion was also significantly higher in the rrp group ( 42.2% vs. 6.3% , respectively ; p<0.001 ) . conversely , wound repairs ( 4.1% vs. 0.2% , p<0.001 ) and extravasation of contrast medium at cystography ( 10.0% vs. 2.1% , p<0.001 ) were more common in the rrp group than in the ralp group , respectively . there was no significant difference in femoral neuropathy ( rrp , 2.1% ; ralp , 2.5% ; p=0.682 ) , postoperative ileus ( rrp , 1.8% ; ralp , 2.3% ; p=0.593 ) , reoperation ( rrp , 0.6% ; ralp , 0.4% ; p=0.664 ) , or postoperative icu care ( rrp , 0.3% ; ralp , 0.6% ; p=0.554 ) rates between the groups . reports from several centers have demonstrated the superiority of ralp over open and laparoscopic prostatectomy with respect to surgical outcomes [ 15 - 17 ] . compared rrp and a robotic approach ( 30 patients in each group ) and reported transfusion rates of 17% and 7% and complication rates of 6% and 6% , respectively . in a similar study , ahlering et al . , comparing the newly introduced ralp with rrp , observed satisfying outcomes in terms of complication rates ( 6.7% vs. 10% , respectively ) . . also showed the superiority of ralp ( 105 patients ) over rrp ( 103 patients ) with respect to postoperative transfusion rates ( 1.9% vs. 14% , respectively ) . however , in the aforementioned studies , multiple surgeons performed the rrp and ralp procedures , and the perioperative complications were not classified by use of the clavien system . compared complications arising from rrp and ralp procedures performed by the same surgeon ; however , the study was limited by a small sample size . we found that the incidence of clavien grade iii - iv complications was significantly lower in the ralp group ( 3.4% ) than in the rrp group ( 7.6% ) . this finding is notable because of the large sample size , the inclusion of a single surgeon series , and the use of the clavien system for classification of the complications . the previously noted advantages of ralp were confirmed in the present study , in which two comparative groups of patients were managed by use of an identical postoperative regimen . the conducting surgeon had abundant experience ( 10 years ) in rrp but had no experience in ralp , or in laparoscopic radical prostatectomy , at the start of the study period . thus , effects of the initial learning curve would be expected to be present in the ralp data . the fact that the complication rates were lower in the ralp group despite the inclusion of a learning curve adds weight to the argument supporting this surgical method in the treatment of prostate cancer . the rate of wound repair after rrp in this study was 4.1% , which was approximately 20 times higher than the rate in the ralp group ( 0.2% ) . the incidences of wound repairs in the united states and europe have been reported to be 0.3% to 3.2% after rrp , whereas two multicenter studies from japan have reported a higher incidence of between 6.0% and 7.5% . the skin incision in rrp is much longer than the short umbilical incision used in ralp for specimen retrieval and this difference might be important when trying to explain the higher incidence of wound repair in the rrp group . although these were not culture - confirmed complications and therefore seroma could not be separated from true wound infections , the more frequent rate of wound infection in the rrp group than in the ralp group could be another possible cause of the higher wound repair rate with rrp . we found that the incidence of extravasation of contrast medium during cystography was higher in the rrp group ( 10.0% ) than in the ralp group ( 2.1% ) . one possible explanation for the lower extravasation rate in the ralp group is that a watertight anastomosis was more likely with the robotic technique owing to better visualization and the use of a running anastomosis . similarly , the urinary retention rate of the rrp group ( 7.0% ) was significantly higher than that of the ralp group ( 2.7% ) . reported that patient- or surgery - specific risk factors for urinary retention were not identified , but they suggested that acute urinary retention develops as the result of edema at the anastomosis . we believe that the running anastomotic suture of ralp might facilitate initial voiding because earlier healing to a watertight state could lead to an earlier decrease in edema . our suggestion is supported by a previous study showing that running anastomotic suture of laparoscopic prostatectomy allowed early catheter removal . considering the duration of hospitalization , our values were far longer in both study arms than those reported in series from the united states , which probably reflected the different pathways of management . specifically , owing to the differences in economic health systems and cultural backgrounds , korean patients often stay in the hospital until the urinary catheter is removed , whereas patients in the united states are usually discharged quickly in the case of an uneventful early postoperative course . comparing transfusion rates from different hospitals can be misleading , because the indication for blood transfusion may vary with clinical praxis , but the transfusion rate of the rrp group in our study was much higher than that reported in other studies . blood transfusion rates of between 3% and 34% have been reported in rrp patient series [ 27 - 29 ] . arai et al . reported that only 6.9% of patients who had predonated an autologous blood subsequently received an allogenic transfusion in an rrp series . by contrast , among the patients who donated no autologous blood before rrp , 45% subsequently received an allogenic transfusion . it is likely that the transfusion rate of our study was high because the patients did not receive autologous transfusion , granulocyte - stimulating factor , or cell saver . moreover , in the early phase of this study , the patients tended to receive transfusion for rapid recovery if their hemoglobin was lower than 10 g / dl after surgery , even though postoperative bleeding was not suspected . however , more recently , patients underwent blood transfusion only when their postoperative hemoglobin was lower than 8 g / dl or had a tendency to decrease . thus , the transfusion rates of the rrp and ralp group were decreased to 18.8% and 0% in the last 50 cases . irrespective of the change in the transfusion rate , the difference in transfusion rate between the rrp and ralp groups was significant in our study . one explanation for the seven times lower transfusion rate in the ralp group ( 6.3% ) than in the rrp group ( 42.2% ) could be a tamponading effect resulting from the pneumoperitoneum , which is beneficial in preventing venous bleeding , the most common source of blood loss during rrp . loss of refinement in the technique of vascular control during rrp may also have contributed to the result . there are challenges in implementing a randomized trial because patients are usually unwilling to be randomly assigned to different treatments and may only accept robotic surgery . however , the clinical characteristics of the patients were not significantly different between the two groups in the present study . this could , of course , have influenced the results , but this must be considered unlikely as an explanation of the difference in complications between these two groups . as previously discussed , the lack of surgical experience with ralp compared with the significant experience with rrp must also be considered when reviewing the outcomes from this study . furthermore , only complications that occurred within 90 days of the surgery were included in this report . late complications such as bladder neck contractures and the need for further surgery for urinary incontinence should be considered in further studies . the introduction of ralp at our institution resulted in a decreased number of patients with urinary retention , extravasation of contrast medium during cystography , and wound repair after surgery . our results show that ralp is associated with a lower complication rate than is rrp , especially the rate of major complications .

purposeto compare the complications of radical retropubic prostatectomy ( rrp ) with those of robot - assisted laparoscopic prostatectomy ( ralp ) performed by a single surgeon for the treatment of prostate cancer.materials and methodsthe postoperative complications of 341 patients who underwent rrp and 524 patients who underwent ralp for prostate cancer at the asan medical center between july 2007 and august 2012 were retrospectively reviewed and compared . complications were classified according to the modified clavien classification system.resultsralp was associated with a shorter length of hospital stay ( mean , 7.9 days vs. 10.1 days , p<0.001 ) and duration of urethral catheterization ( 6.2 days vs. 7.5 days , p<0.001 ) than rrp . major complications ( clavien grade iii - iv ) were less common in the ralp group than in the rrp group ( 3.4% vs. 7.6% , p=0.006 ) . there were no significant differences in medical complications between procedures . considering surgical complications , urinary retention ( 7.0% vs. 2.7% , p=0.002 ) and wound repair ( 4.1% vs. 0.2% , p<0.001 ) were more common after rrp than after ralp . extravasation of contrast medium during cystography was more common in the rrp group than in the ralp group ( 10.0% vs. 2.1% , p<0.001).conclusionsralp is associated with a lower complication rate than rrp .

INTRODUCTION MATERIALS AND METHODS 1. Patients 2. Surgical techniques 3. Evaluation of complications 4. Statistical analysis RESULTS DISCUSSION CONCLUSIONS

PMC4727650

telemedicine can be defined as the use of telecommunication technologies to provide medical information and service . historically , it started more than 4 decades ago to allow communication and discussion between physicians of small hospitals and those of large tertiary care hospitals for non - acute cases where advanced treatment must be planned and agreed on . therefore , telemedicine started in an asynchronous module where data can be stored and forwarded and then reviewed offline . however , acute care has developed significantly in the last few decades as well as telecommunication technologies , which made synchronous telemedicine possible and in a different format from telephone - based to audio - video teleconference , and this may go further with the attachment of peripheral devices for robotic surgery or procedures under tele - guidance . we can define telestroke as the emergency use of telecommunication technologies to provide appropriate acute stroke therapy at a distance . a telestroke program using telephone consultation is cheap and takes less time to initiate and to complete , however , it is associated with significant limitations and the risk of making a wrong diagnosis or at least inaccurate acute stroke related deficit evaluation , and then the wrong thrombolysis decision . most of the current telestroke programs use synchronous two - way audio - video teleconferences , and this is our area of interest in this article . the patient is clinically evaluated using the national institute of health stroke scale ( nihss ) and the brain image is reviewed by a stroke neurologist from a distance , and then a thrombolysis decision is taken . in one randomized controlled study , the audio - video teleconference was compared to the telephone - based telestroke and was found to be more sensitive , more specific , and more likely to lead to the right thrombolysis decision.3,4 after confirming the diagnosis of acute stroke , there are 2 important measures to increase the recanalization rate with significant clinical benefit : thrombolysis rate ( percentage of acute stroke patients who received iv t - pa within the therapeutic window ) and iv t - pa needle time ( time from stroke onset to iv t - pa bolus ) . currently , 2 options exist for acute stroke patients care in medically under served areas : either urgent patient transfer to a tertiary care hospital where acute stroke treatment can be administered , or managing the patient at the local hospital . patient transfer can be via ambulance or air evacuation , which is associated with high transportation costs , smaller increase in thrombolysis rate , and smaller chance for significant recovery if they were treated , because most likely the iv t - pa needle time will be within the last third of the therapeutic time - window , which is associated with higher number needed to treat.5 the second option necessitates telestroke technology so stroke experts share with local physicians the duty of applying evidence - based acute stroke therapy . we need to look here for 2 measures , the validity of patient assessment using the nihss through teleconference , and the effectiveness , including success , and complication rate , of telestroke based intravenous thrombolysis . few studies have looked into the accuracy of patient assessment using the nihss . in one study,6 the nihss scores of 20 patients were compared to their nihss scores carried out remotely through a broadband - connected workstation . both assessments were carried out by qualified neurologists . paired t tests and pearson correlation coefficients were very strong ( r=0.9552 , p=0.0001 ) indicating that high scores in bedside correlate with high scores remotely . for the second measure , we will review published data from major telestroke programs in europe and north america . the reach telestroke network started in 2003 in georgia , usa.7 by 2005 , the network had 7 hospitals in rural areas connected to the medical college of georgia via one - way video and two - way audio teleconference system . between march 2003 and may 2005 , 194 acute stroke consultations were made within the 3-hour therapeutic window and 30 patients with a median nihss of 12.5 were treated with iv t - pa . rapid improvement defined as a drop of 4 or more nihss points within 24 hours occurred in 18 out of 30 treated patients , and no symptomatic intracerebral hemorrhage occurred . the study interestingly showed a significant reduction in onset to treatment after one year of operation from 143 to 111 minutes . the most common reasons for not treating were rapid improvement , nihss was below 4 , beyond therapeutic window , or presence of seizure or hemorrhage prior to treatment . some other data recently published represents the experience of the university of pittsburg medical center telestroke network with its 12 spoke facilities through audio - video conferencing equipment.8 between july 2008 and december 2009 , 351 telestroke consultations were performed . eighty - three stroke patients received iv t - pa at the spoke hospitals , and 59 patients at the hub center . the 2 groups were similar in their baseline characteristics including median nihss scores and mean onset to treatment time . the mean arrival to treatment time was longer in the telestroke patients ( 67.8 versus 98.9 minutes ) . the study showed no statistical significant difference between the 2 groups in favorable outcomes , symptomatic intracerebral hemorrhage , and mortality rates . the northern alberta telestroke service started in 2003.9 by 2007 , 7 spoke centers were connected to the university of alberta hospital ( 4 via audio - video teleconference , and 3 via telephone only ) . in their first 2 years of experience , the telestroke was activated 210 times and only 44 patients were candidates for thrombolysis using iv ten ( 23% ) of them were assessed by the er physician at the spoke centers without video assessment by a stroke neurologist . sixteen ( 40% ) patients had a modified rankin scale less than 2 by 90 days from iv t - pa , and 9 deaths occurred within 90 days from iv thrombolysis . only 2 ( 4.5% ) of the treated patients had t - pa complications with intracerebral hemorrhage , and the rest died due to other mechanisms . the telemedical project for integrative stroke care ( tempis ) was established in 2003 serving a large area in south east bavaria , germany.10 it comprised 12 spoke hospitals connected to 2 academic centers in munich via video teleconference and ct / mri image transfer . currently , tempis is considered to be the largest telemedical stroke service in the world . tempis was associated with significant improvement of acute in - hospital stroke treatment in terms of quality indicators such as rapid brain imaging , thrombolysis rate , assessment of swallowing disorders , and early rehabilitation therapy.11 when patients served with tempis were compared to similar acute stroke patients within the same region , but not served with telemedicine , tempis showed significant reduction in death and dependency outcome at 3 , 12 , and 30 months,10,12 without a significant difference in mortality rate alone . the thrombolysis rate in most of the published data of different telestroke networks ranges from 18 - 51%.7 - 10 reasons for no iv t - pa include stroke onset beyond 270 minutes , presence of contraindication that was not recognized by the er physician at the spoke hospital , or patient having a stroke mimic like seizure with todd s paralysis , cns neoplasm with sudden deterioration , cns infection , and others . considering the threat of these acute stroke mimics , on - call stroke neurologists are still of great benefit to the patients and the spoke hospitals . the small waste of telestroke resources on acute stroke mimics is well compensated by the gain of managing these urgent neurological conditions despite the fact that some patients were required to be transferred to the university hospital anyway . with time and experience there are 3 nonexclusive methods of clinical operation of telestroke : drip and keep , which means after telestroke consultation and approval for iv t - pa , the iv t - pa is infused at the spoke center followed by admission there for observation and stroke work up . this requires a local stroke unit with at least minimal acceptable quality of care with multidisciplinary teams to have an integrative telestroke network . a follow up assessment by a stroke neurologist can also be carried out by telestroke , or through outpatient visit to the hub center a few weeks later . for further complications during the acute phase of treatment , urgent telestroke consultation can be initiated again by the treating physician at the hub hospital . the second method is the ship method , which is recommended for patients with an acute stroke with contraindication for iv t - pa , but that can still have intraarterial t - pa , or an endovascular procedure , and mechanical removal / disruption of the clot ( for example : patients on anti - coagulation with inr > 1.6 ) . here , the patient gets transferred directly to the hub center for urgent treatment and intervention . the third way is drip and ship and indicated for those acute stroke patients who are candidates for iv t - pa . however , the consulting spoke hospital can not provide appropriate post - tpa care or stroke workup . the patient can have the iv t - pa infused locally , and then shipped to the hub center . developing telestroke networks may better encourage more spoke centers to participate if they choose drip and ship since it put less load on the spoke centers , and with time and experience , spoke centers can start admitting patients after thrombolysis . it is also important to mention that the type of clinical practice can be influenced by financial incentives and reimbursements for physicians and hospitals , and by clear job descriptions and duties at each end of the telestroke network , in case of complications or medico - legal claims . the quantity and type of manpower required for a telestroke network varies , and depends on the proposed size of the network , type of clinical operation , and complexity of care . based on experience , each hub center needs 4 or more stroke neurologists , one of them assigned as the medical director of the network , one telestroke manager , one data manager , 2 telestroke nurses , and 2 information technologists , plus the clinical and administrative back up support from the tertiary hospital . for each spoke center , you need to involve at least the emergency physicians , emergency nurses directed by the head of er there , plus a program manager . for drip and keep practice , the multidisciplinary teams including the internists should be added . effective and interactive relationships between different network sites , frequent sites visits between hub and spoke personnel , continuing medical educational activities , and sharing data , and successful stories are mandatory for sustainability and further development . the recommended quality and performance measures for acute stroke units should be used to assess quality and performance of the telestroke networks plus other measures that are specific to telestroke , such as those concerned with communication ( table 1 ) . the director of the telestroke network must obtain the data regularly from each spoke , and hub center , and discuss them at least once annually with all network staff in a transparent fashion with the intention to improve quality and reduce failures . professionals of each telestroke network should also compare their network performance with other networks nationally and internationally . today , most tertiary care hospitals are either practicing some form of telemedicine or considering it soon . the practice can be limited to certain health care networks of similar financial and legal jurisdiction , or may go beyond different states borders . therefore , the first legal issue is getting the license from an appropriate body based on the defined map of telemedicine practice , and making sure the practice does not exceed that map . the second challenge is staff accreditation as different hospitals may have different regulations and requirements . for example in saudi arabia , although the saudi commission for health specialties has a set of regulations concerning staff accreditation to designated positions , tertiary care hospitals like king khalid university hospital have added further requirements . therefore , mutual agreement on staff accreditation between the spoke and hub centers is needed with defined rules and areas of practice , and then hospitals do not need to change their accreditation requirements in order to practice telemedicine . the third challenge is agreement on sharing electronic medical records and patient s data without breaking rules of confidentiality and privacy . this agreement can be initiated by the hospitals boards , but needs to be approved by the appropriate licensing / legal body . as the telestroke network expands and more cases are treated , the hub stroke neurologists may become overwhelmed between their regular hospital duties and telestroke cases , but they will still be legally responsible for delivering the telestroke service on time ; otherwise , they may be blamed for negligence . the director of the telestroke network needs to adequately estimate the required number of stroke neurologists based on the size of the population served , and ensure a fair and stable reimbursement system ; otherwise , staff - recruitment failure may occur . a medical error or failure to serve the acute stroke patient on time can be related to the staff at the spoke center , staff at the hub center , technology related , patient related , or mixed . therefore , a 24/7 documentation and auditing system is needed . the job description of every staff member within the network must be clearly written and defined . regular telestroke morbidity and mortality educational activities must run and be shared between all network sites to enhance the learning curve at different levels as well as discussing successful stories . for example in the usa alone , the direct and indirect costs associated with stroke for 2008 are approximately us$65.5 billion.13 fagan and colleagues14 showed that acute stroke treatment with iv t - pa is associated with an incremental cost savings of us$8,000 per qaly gained , and telestroke was proved to increase thrombolysis rates mainly in patients living in suburban and rural areas.15 there are 2 cost categories associated with telestroke : equipment costs and telestroke running costs , including physician s compensations and salaries for network employees . the annual telestroke hub equipment cost is approximately us$16,204/site , and the annual telestroke spoke equipment costs are approximately us$5,309/site,16 which are cheap when we consider today s medical costs . when looking into the cost - effectiveness of telestroke , we consider the telestroke running costs from one side , which are more or less predictable per network s size , versus the thrombolysis - related savings , such as less or no disability , shorter length of stay at hospital , and savings from less patients transportation . the cost - effectiveness ratio can be non - constant and affected by the network s performance and number of cases treated . too reduce its costs further , it can be part of a synchronous telemedicine network so some resources are shared with other acute medical consultation services . in conclusion , telestroke is the only solution in many countries to serve suburban areas and to increase the thrombolysis rate . new regulations are needed to ensure its legality and stability , and initiatives should come from stroke neurologists , er physicians , and medical directors of small hospitals .

despite developments in acute stroke therapies , stroke continues to be a leading cause of death and disability worldwide . one major limitation from intravenous thrombolysis with tissue plasminogen activator ( t - pa ) is patient s arrival to the emergency room at a tertiary care hospital after the therapeutic time - window , which is generally 270 minutes . the problem is worse for people living in suburban areas where stroke expertise can be missing . therefore , telestroke networks were developed at several sites where a stroke neurologist at a tertiary hospital participates through synchronous audio - video teleconference in confirming diagnosis of stroke , assessing risks and benefits of giving iv t - pa , and making the decision with the patient and emergency physician at the local hospital . in this article , we will review the experience of major telestroke networks in north america and europe , and the evidence of its safety and cost - effectiveness . telestroke complexity , with regard to practice , manpower , quality assurance , and legal issues will be discussed briefly .

Telemedicine Telestroke Effectiveness of telestroke Three different methods of practicing telestroke Manpower for a successful telestroke network Telestroke quality measures Legal issues Economics

PMC5170848

born and raised in italy , i worked as an undergraduate assistant in milan , studying dna replication in budding yeast . the lab was small and tight - knit , and the work of undergraduate students was valued and rewarded . energized by this experience , i decided to move edinburgh to pursue my dream : being a full - time researcher . i did nt know exactly what i wanted to do , everything sounded exciting , but understanding the organization and stability of genomes particularly intrigued me . an opportunity arose to work in a basic biology laboratory that studied chromatin , so , in 1998 , i joined the group of robin allshire at the medical research council human genetics unit in edinburgh , scotland , for my doctoral work . i ll never forget the time i was walking to the lab from my apartment in the lovely comely bank neighborhood on yet another rainy day . out of the blue , i heard a strange crashing noise coming from the road , the sound of something large and soft hitting the cobblestones . a swan had attempted to land on the slick , shiny , wet surface of the road , mistaking it for water . my apartment was near a park with a swan pond , and this was the second time i had to call the local police and wait for them to come and rescue an injured bird , warning drivers to slow down and maneuver carefully around it . the police were accustomed to getting these calls from residents in the area , but when i called the lab to let people know i was running late , yet again because of a fallen swan , my lab mates would nt stop laughing . it was a very exciting time for chromatin biology , as the importance of novel histone modifications was just beginning to emerge . my project focused on understanding the chromatin requirements for the silencing of pericentric heterochromatin in the fission yeast . because of previous work in budding yeast , we had predicted that silencing mechanisms in other organisms would be mediated by the hypoacetylation of the histone h4 n - terminus . i had just embarked on mutating individual histone h4 lysines when thomas jenuwein s lab discovered that mouse suvar39h had histone h3 lysine 9 methyltransferase activity ( rea et al . , 2000 ) . knowing that suvar39h proteins are required for silencing in the fission yeast saccharomyces pombe and flies , i quickly shifted my attention to histone h3 and showed that mutating lysine 9 completely abolished silencing , providing direct evidence that this residue is the key target of this family of methyltransferases and highlighting the remarkable resemblance between the heterochromatin of s. pombe and that of more complex organisms ( mellone et al . , 2003 ) . seeing how much we understand today about heterochromatin and silencing , i cherish having been part of the early work and marvel at how much progress the field has made since then . working with robin allshire taught me to be critical of my data , to connect concepts in a deep and mechanistic manner , and to think about my experiments in the context of the bigger picture . learning to write about my work in a different language felt overwhelming , but he painstakingly edited my thesis and papers , showing me the importance of editing and revisions . working on a basic biology problem was exciting , and the broad implications of the potential findings motivated me . i also realized how much we did nt know about the basic principles of chromatin dynamics and their impact on chromosome inheritance . toward the end of my phd work , i became interested in the centromere - specific histone h3 , called cenp - a , which had been hypothesized to epigenetically i performed experiments that manipulated both canonical histone gene dosage and centromere size , and we determined that cenp - a chromatin was plastic , that is , it had the ability to spread and shrink ( castillo , mellone , et al . , 2007 ) . my exposure to fluorescence microscopy in the allshire lab made me realize how much i loved being able to visualize the structures that fascinated me most : chromosomes and mitotic spindles . the idea that centromeres might be defined epigenetically through the presence of cenp - a , rather than by specific dna sequences ( karpen and allshire , 1997 ) , got me completely hooked on centromeres . i became very interested , perhaps even obsessed , with understanding how cenp - a becomes specifically localized to centromeres and maintained there across cell generations . for my postdoc , i decided that i wanted to study this process using a genetic system that would also afford beautiful cytology . in 2003 , i joined the laboratory of gary karpen , which uses drosophila to study centromeres and heterochromatin , at the lawrence berkeley national laboratory and the university of california , berkeley . the novel high - throughput rna interference ( rnai ) technology that was being developed by norbert perrimon at the drosophila rnai screening center at harvard seemed like an ideal entry point to identify the largely unexplored regulators of centromere chromatin assembly using a cell - imaging screen . this collaborative genome - wide rnai screen ( which involved sylvia erhardt along with myself in the karpen lab and craig betts in aaron straight s lab at stanford ) identified critical factors of centromere function at a time when very little was known about the cenp - a deposition pathway . these factors included centromere - associated proteins and cell cycle regulators ( erhardt , mellone , betts , et al . , 2008 ) , which i was able to later pursue in my own research group . he gave me freedom and responsibility ; supported me unconditionally through two maternity leaves ; and worked closely with me on developing grants , papers , and research seminars . his relentless encouragement and faith in my abilities were instrumental in my decision to seek a faculty position . in 2009 , my husband , two young daughters , and i uprooted ourselves to move to connecticut , the land of broccoli fields , as my good friend from italy calls it , due to the appearance of its tree - covered landscape from an airplane . the hilarious photo on my university of connecticut i d , taken on my first day as a faculty member , captured my initial emotions : is this really happening ? what if i ca nt do this ? the initial few months were challenging ; the gorgeous old university campus was a maze of brick buildings and trees , and i kept getting lost . at uconn i was once again surrounded by large birds , but instead of swans , it was the plentiful canada geese that pass through campus every year in the thousands . i missed lunches with my fellow postdoc friends from berkeley , needing a good laugh or to commiserate about difficult experiments . fortunately , it was nt long before i mastered my new campus s geography and made new friends , both at uconn and outside work . while it seemed i had less time to socialize , i soon found colleagues and friends who supported me and laughed with me . when i started my lab , our understanding of the mechanisms of centromeric chromatin assembly was very limited . as a first step in my independent research program , i followed up on experiments started in the karpen lab and elucidated the functional cell cycle dynamics of cenp - a and its effectors from our rnai screen cyclin a , cal1 , and cenp - c ( mellone et al . , 2011 ) . an inherent challenge of dissecting the centromere assembly pathway is that a mutation or knockdown of centromere components often results in centromere disruption and subsequent cellular lethality . to overcome this , we used genomic engineering in drosophila s2 cells , implementing an inducible tethering system ( laci / laco ) to dissect the stepwise formation of cenp - a chromatin without compromising viability . this was critical in providing the first experimental evidence that cal1 is a cenp - a assembly factor required for centromere establishment ( chen , dechassa , et al . , 2014 ) . our studies showing that centromeric chromatin can be established de novo in flies are consistent with the occurrence of neocentromeres ( centromeres that form at new locations ) in humans . but many fundamental questions remain : how are canonical nucleosomes reorganized to make room for cenp - a during its deposition ? are cells able to detect the presence of a spurious centromere and to inactivate it ? recent graduate students in my lab have taken on some of these challenges , showing that cenp - a deposition is directly coupled to and requires transcription ( chen et al . , 2015 ) and that cenp - a evolves in concert with its chaperone ( rosin and mellone , 2016 ) , but much exciting work remains to be done not only to inform mechanistic models for centromere specification but also to explore the genomic landscape and evolution of these fascinating chromosomal structures . as with most new science faculty , i have experienced the ups and downs of staffing a functional laboratory , obtaining funding , publishing , and teaching effectively . in fact , many times i felt like those swans in edinburgh confused , disappointed , and alone but just like those swans , who ultimately made it safely back to their pond ( in one case after some rehabilitation ) i have always had somebody to turn to for help and support when things did nt go as planned ( by the way , they rarely do ) . despite the highs and lows , doing research for a living remains a highly rewarding and creative career that offers both great flexibility ( especially helpful when also raising a family ) and the fortunate opportunity to encounter new ideas , meet new people , and travel to new places along the way .

it is an honor to become a part of the talented group of cell biologists who have received this award before me . while running a research group certainly has its ups and downs , i love being a faculty member and am continuously excited by the prospect of scientific discoveries yet to be made . i have benefited from the support of many people over the years and hope to be able to do the same for others through my mentoring and teaching .

SKYDIVING SWANS AND CENTROMERES BROCCOLI FIELDS AND NEW CHALLENGES FINAL THOUGHTS

PMC3795899

while macroautophagy , herein simply termed autophagy , plays an indispensible role in the intracellular degradation of proteins and organelles , there is growing evidence that also implicates this process in protein secretion . this link includes a role for autophagy in regulating unconventional secretion of acb1 in yeast and interleukin-1 ( il-1 ) in mammalian cells . for instance , the constitutive secretion of il-6 and il-8 from senescent cells appears to involve the formation of a specialized cellular domain termed the tor - autophagy spatial coupling compartment ( tascc ) . this compartment is directly adjacent to the trans golgi network and is enriched for both signaling proteins such as mtor , as well as for autophagic vacuoles . finally , there are a growing number of studies suggesting a connection between components of the autophagy machinery and the regulated secretion of intracellular lysosomes or granules . for instance , deficiency in specific essential autophagy proteins appear to impair regulated secretion from the intestinal paneth cell , bone marrow derived mast cells , pancreatic -cells , melanocytes , osteoclasts and vestibular epithelial cells . exocytosis from endothelial cells represents one of the first lines of defense following vascular injury . specific secretory granules within the endothelial cell known as weibel - palade bodies ( wpbs ) contain numerous biologically active molecules , although von willebrand factor ( vwf ) is by far the most abundant . indeed , vwf is required for formation of wpbs , and heterologous expression of vwf in non - endothelial cells can induce the formation of wpb - like structures . once secreted , vwf multimers can assemble into long strings that can become tethered to the underlying connective tissue while simultaneously ensnaring circulating platelets . impaired production , processing or secretion of vwf results in the clinical syndrome known as von willebrand disease , recognized as the most common inherited bleeding disorder . the production of wpbs is a complex process involving dimerization and disulfide bond formation in the endoplasmic reticulum followed by furin - dependent cleavage in the golgi complex . the vwf multimers that form can then be folded , in a ph - sensitive fashion , into tubular structures . while structural mutations in vwf can lead to defects in multimer formation or tubulation , relatively little is known about what other cellular processes regulate the number , ph or secretory potential of wpbs . here , we demonstrate that autophagy plays a critical role in the biology of wpbs and regulates the in vitro and in vivo release of vwf . while there is a growing understanding of the molecular and biological role of autophagy , the role this process plays within the vasculature is poorly understood . in an effort to better understand how autophagy might contribute to vessel homeostasis , we analyzed electron micrographs of primary human umbilical vein endothelial cells ( huvecs ) . both wpbs and autophagosomes have distinctive appearance by electron microscopy , with the former exhibiting a classical cigar shaped appearance and the latter being an intracellular organelle often filled with diverse cellular contents surrounded by a unique double membrane structure . surprisingly , we noted that within endothelial cells , wpbs and autophagosomes were often found in close proximity . moreover , we noted multiple instances in which a wpb appeared to be very near and actively fusing with an autophagosome ( fig . 1a and supplemental fig . examination of the contents of autophagosomes also revealed the presence of what appeared to be wholly or partially intact wpbs , clearly identifiable by their unique shape and internal striations running parallel to their long axis ( fig . 1b ) . to further pursue these observations we performed additional immunogold labeling coupled with electron microscopy analysis to assess the distribution of vwf within endothelial cells . these studies revealed that as expected , vwf was present at high abundance within mature wpbs ( fig this immunogold labeled vwf signal appeared to be composed of both free protein , as well as , what appeared to be wholly or partially intact wpbs ( fig . 1d and supplemental fig . 1c - e ) . analyzing 30 random section containing several hundred autophagosomes revealed that 17 % of the autophagosomes had no detectable vwf signal ( 48/279 ) , 42% ( 117/279 ) of the autophagosomes had a moderate amount of vwf ( 1 - 10 immunogold particles ) and 41% ( 114/279 ) of the autophagosomes had a robust signal for vwf ( > 10 immunogold particles per autophagosome ) . the electron microscopic observations suggested that autophagosomes , and by extension autophagy , might somehow modulate vwf levels or secretion . autophagosome formation requires that the cytosolic form of lc3 ( lc3-i ) be conjugated in an atg5 and atg7 dependent fashion to form the lipidated lc3-ii species . based on these past observations , we next sought to analyze the distribution of vwf and lc3 ( atg8 ) using super resolution structured illumination microscopy . these images revealed the expected cigar shaped wpb morphology and the classical spherical punctae of lipidated lc3-ii ( fig . 1e ) . within endothelial cells , super resolution microscopy revealed that endogenous vwf and lc3 were clearly separable . nonetheless , at any given time , a substantial fraction of wpbs were closely associated with a lc3 signal ( 37/224 wpbs or 16.7% ) with a smaller percentage of wpbs ( 7/224 or 3.2% ) appearing to be surrounded by a structure consistent with a lc3-coated autophagosome ( fig . 1e and supplemental movie 1 ) . a separate analysis using confocal microscopy came to a similar conclusion , namely that a substantial fraction ( approximately 30% ) of intracellular lc3 and vwf spots had a centroid to centroid distance of less than 1.5 m ( supplemental fig . finally , using two color confocal live cell imaging , we could capture in real time , the apparent direct interaction of a lc3-gfp autophagosome with a vwf - mcherry positive wpb ( fig . nonetheless , additional dual - color ( lc3-mcherry and vwf - gfp ) or single color ( lc3-gfp ) tirf imaging suggested these interactions were rare and that under normal circumstances , autophagosomes do not appear to directly fuse with the plasma membrane ( supplemental movie 2&3 ) . to begin to address whether autophagy modulates vwf biology , we performed lentiviral shrna mediated knockdown of atg7 in human endothelial cells . in atg7 knockdown cells , western blot analysis confirmed a significant reduction in atg7 expression ( fig . assessment of autophagic flux often relies on the simultaneous measurements of the level of expression of the multidomain , signaling adaptor protein p62/a170/sqstm1 , hereafter referred to simply as p62 , along with the ratio of lc3-ii to lc3-i . a rise in p62 levels and a fall in lc3-ii are consistent with impaired autophagic flux . as expected , knockdown of the essential autophagy gene atg7 had the expected effects on these well characterized markers of autophagic flux ( fig . we then asked whether knockdown of atg7 altered the stimulated release of vwf . using an elisa - based method of the culture medium , we detected an increase in vwf secretion following stimulation with histamine or vegf ( fig . we saw similar effects on vwf secretion using an alternative and independent method for knockdown of atg7 that employed sirna ( supplemental fig . the reduction in the stimulated release of vwf was further confirmed by direct western blot analysis of the endothelial cell supernatant ( fig . 2c , d ) . a recent report has suggested that disruption of autophagy might alter intracellular calcium signaling . given the central role of calcium in stimulated exocytosis from the endothelium , we wondered whether such a mechanism might explain our observations . using a calcium sensitive fluorometric probe , we could not however detect significant differences in ligand - induced calcium transients between control and atg7 knockdown cells ( fig . similarly , other proximal signaling events such as extracellular regulated kinase ( erk ) activation appeared to be unaffected by atg7 knockdown ( supplemental fig . 4 ) . we next sought to establish whether other genetic or pharmacological inhibitors of autophagy had similar effects . knockdown of the essential autophagy gene atg5 alone or in combination with atg7 also inhibited vwf release ( fig . following histamine stimulation , the combined knockdown of atg5 and atg7 appeared slightly more effective than single knockdown of either atg7 or atg5 in blocking vwf secretion ( fig . incubation of human endothelial cells with chloroquine or bafilomycin , two known inhibitors of autophagic flux , also resulted in a marked inhibition of ligand - stimulated vwf secretion ( fig . in contrast , knockdown of atg5 or atg7 did not appear to alter ligand - stimulated release of endothelin-1 from endothelial cells ( supplemental fig . 5b ) . in addition , while knockdown of atg6 ( beclin 1 ) was quite effective in reducing atg6 expression ( fig 2i ) , it had only a very modest effect on markers of autophagic flux ( supplemental fig . we next sought to attempt to get a better understanding of why knockdown of an essential autophagy gene such as atg7 might inhibit the secretion of vwf . following synthesis , vwf undergoes a series of processing events in the er and golgi that eventually lead to maturation of the protein and incorporation into wpbs . using a quantitative elisa - based method we determined that the total intracellular level of vwf protein was not significantly different between control and atg7 knockdown endothelial cells ( fig . similar results were observed following knockdown of atg5 or combined atg5 and atg7 knockdown ( supplemental fig . we next sought to analyze whether differences might exist in the processing , maturation and secretion of vwf . the processing of vwf involves a furin - dependent proteoltyic cleavage within the trans - golgi network with the pro - vwf precursor distinguishable from mature vwf based on electrophoretic mobility . we observed that the steady state ratio of pro - vwf to mature vwf was significantly altered in atg7 or atg5 knockdown cell ( fig . consistent with a defect in vwf processing , we also noted a reduction in the number of mature wpbs observed in atg7 knockdown cells ( fig . we saw similar reduction in wpbs in endothelial cells with either atg5 knockdown or following pharmacological inhibition of autophagy using chloroquine or bafilomycin ( supplemental fig . in contrast , knockdown of atg6 did not alter the number of mature wpbs ( supplemental fig . these results suggest that one explanation for our observation with atg5 or atg7 knockdown is that these strategies , while not altering overall vwf expression , appear to inhibit the processing and maturation of vwf into mature wpbs . the formation of wpbs is believed to occur at the trans - golgi network ( tgn ) and autophagy is known to be critical for the homeostatic maintenance of both the er and golgi . furthermore , the ph of wpbs is known to be acidic and this low ph is essential for the shape of the wpbs , as well as vwf tubulation and function . using previously described methods , we were able to establish that wpbs within atg7 knockdown cells exhibited a higher ph than wpbs found in control cells ( fig . 4a and supplemental fig . consistent with previous results , when assessed by confocal microscopy , this higher ph in atg7 knockdown cells resulted in rounder and less rod - shaped wpbs ( fig . this also translated into a statistically significant reduction in the length of wpbs found in atg7 knockdown cells ( fig . similar observations were evident by electron microscopy where we observed that knockdown of atg7 resulted in wpbs that were shorter , rounder and had less distinct tubulation ( fig . whether in addition to a processing defect , there was also an effect of atg7 on the secretion of mature wpbs , we employed a previously described system using live cell imaging of the secretion of a vwf - gfp chimeric fusion protein . expression of this tagged vwf results in assembly of wpbs containing the co - expressed fluorescently labeled vwf protein ( supplemental fig . consistent with the alterations in processing described above , the number of gfp - positive wpbs was reduced in cells where atg7 was knockdown ( supplemental fig . 9b ) and the wpbs that formed , had an altered , more rounded morphology ( supplemental movies 4 , 5 ) . using this system , in conjunction with total internal reflection fluorescence ( tirf ) microscopy , we could monitor individual secretion events of gfp - positive wpbs that localized near the plasma membrane ( fig . our analysis demonstrated that knockdown of atg7 resulted in reduced wpb secretion after histamine stimulation ( fig . 4 g ) . in an effort to extend these observations to an in vivo situation , we conditionally deleted atg7 within endothelial cells by crossing mice harboring atg7 floxed alleles with transgenic mice expressing the previously described ve - cadherin cre recombinase . parental mice both of which bore the genotype atg7 ; ve - cadherin cre were crossed to generate offspring that were either atg7 ; ve - cadherin cre ( hereafter denoted as atg7 ) or atg7 ; ve - cadherin cre ( hereafter denoted as control ) . given the relatively small contribution of endothelial cells to overall organ mass , we did not observe a significant reduction in atg7 expression in the tissues and organs isolated from atg7 mice ( supplemental fig . primary cultures of mouse endothelial cells isolated from atg7 mice exhibited the expected reduction in atg7 expression ( fig . the residual expression of atg7 in these cells may represent a combination of incomplete deletion with ve - cadherin cre , as well as potentially some degree of contamination of non - endothelial cells in these primary cultures . nonetheless , this level of atg7 expression was sufficient to induce a reduction in autophagic flux as evidenced by the lc3-i / ii ratio and p62 levels ( fig . similar to our observations employing knockdown of atg7 in human endothelial cells , we did however observe a reduction in the number of mature wpbs in endothelial cells derived from atg7 mice ( fig 5b ) . while conditional deletion of essential autophagy genes in tissues such as brain led to marked alterations in tissue homeostasis and early mortality , atg7 mice exhibited no obvious vascular phenotypes . similarly , atg7 mice exhibited no increase in mortality over the first year of life ( unpublished observations ) . assessment of vascular density demonstrated that atg7 mice were indistinguishable from control animals ( fig . 5c ) . in addition , while autophagy has been implicated in angiogenesis , we saw no consistent or reproducible abnormalities in atg7 mice when post - natal retinal angiogenesis was assessed ( fig . furthermore , the endothelium and underlying architecture of large vessels were similar in atg7 and control mice ( fig . we also observed no reduction in the level of vwf expression in atg7 mice either by immunohistochemical ( fig . we next asked whether atg7 mice exhibited defects in vwf secretion . in control mice , 6a , fold increase : 1.5 + / 0.04 , n=27 mice , p<0.001 pre versus post ) . this increase in vwf in response to subcutaneous epinephrine injection is similar in magnitude to previous reports . in contrast , atg7 mice failed to respond in a significant fashion to an epinephrine challenge ( fig . 6a , fold increase : 1.17 + / 0.05 , n=13 mice , p = ns pre versus post ) . this lack of stimulated epinephrine release was also evident in mice where atg5 had been conditionally deleted again using ve - cadherin cre . similar to atg7 mice , atg5 mice also failed to respond to epinephrine ( fig . 6a , fold increase : 1.13 + / 0.07 , n=9 mice , p = ns pre versus post ) . neither atg5 mice nor atg7 mice had a significant reduction in basal vwf ( fig . similarly , basal levels of circulating p - selectin levels were not altered between control and atg7 mice ( supplemental fig . 12 ) . to therefore further assess the role of autophagy in regulating basal vwf levels , we analyzed mice having a complete , whole body knockout of atg7 . previous studies demonstrated that these total body atg7 knockout mice rarely survive for more than 48 hours after birth . when we analyzed atg7 mice shortly after delivery , we did note a reduction in plasma vwf when compared to wild type littermates , perhaps reflective of a more complete knockout in this model then can be achieved using conditional approaches ( fig . we next sought to further characterize the physiological importance of atg7 in vwf processing in vivo . consistent with the observed in vitro deficit in vwf processing , we noted that on non - reducing gels , plasma vwf analyzed from atg7 mice contained diminished levels of the biologically active , higher molecular weight multimers ( fig . consistent with this reduction in high molecular weight vwf , coupled with the decrement in stimulated vwf release ( fig . 6a ) , we noted a significant increase in measured bleeding time when control mice were compared to atg7 mice ( fig . such differences occurred in the absence of any measurable differences in various hemostatic parameters ( supplemental fig . furthermore , we did not see any obvious abnormalities in platelet morphology or platelet vwf levels that could have potentially contributed to the observed alterations in the measured bleeding time ( supplemental fig . 14 ) . given that one of the many effects for chloroquine is inhibition of autophagy and since this agent is approved for human usage , we next randomized mice to saline or chloroquine ( cq ) injections . after 9 days , basal plasma vwf levels in cq treated and vehicle treated mice were similar ( supplemental fig . nonetheless , direct assessment of hemostasis revealed that similar to atg7 mice , pharmacological inhibition of autophagic flux resulted in a significant prolongation of bleeding time ( fig . 6e and supplemental fig . while there is a growing understanding of the molecular and biological role of autophagy , the role this process plays within the vasculature is poorly understood . in an effort to better understand how autophagy might contribute to vessel homeostasis , we analyzed electron micrographs of primary human umbilical vein endothelial cells ( huvecs ) . both wpbs and autophagosomes have distinctive appearance by electron microscopy , with the former exhibiting a classical cigar shaped appearance and the latter being an intracellular organelle often filled with diverse cellular contents surrounded by a unique double membrane structure . surprisingly , we noted that within endothelial cells , wpbs and autophagosomes were often found in close proximity . moreover , we noted multiple instances in which a wpb appeared to be very near and actively fusing with an autophagosome ( fig . 1a and supplemental fig . examination of the contents of autophagosomes also revealed the presence of what appeared to be wholly or partially intact wpbs , clearly identifiable by their unique shape and internal striations running parallel to their long axis ( fig . 1b ) . to further pursue these observations we performed additional immunogold labeling coupled with electron microscopy analysis to assess the distribution of vwf within endothelial cells . these studies revealed that as expected , vwf was present at high abundance within mature wpbs ( fig this immunogold labeled vwf signal appeared to be composed of both free protein , as well as , what appeared to be wholly or partially intact wpbs ( fig . 1d and supplemental fig . 1c - e ) . analyzing 30 random section containing several hundred autophagosomes revealed that 17 % of the autophagosomes had no detectable vwf signal ( 48/279 ) , 42% ( 117/279 ) of the autophagosomes had a moderate amount of vwf ( 1 - 10 immunogold particles ) and 41% ( 114/279 ) of the autophagosomes had a robust signal for vwf ( > 10 immunogold particles per autophagosome ) . the electron microscopic observations suggested that autophagosomes , and by extension autophagy , might somehow modulate vwf levels or secretion . autophagosome formation requires that the cytosolic form of lc3 ( lc3-i ) be conjugated in an atg5 and atg7 dependent fashion to form the lipidated lc3-ii species . based on these past observations , we next sought to analyze the distribution of vwf and lc3 ( atg8 ) using super resolution structured illumination microscopy . these images revealed the expected cigar shaped wpb morphology and the classical spherical punctae of lipidated lc3-ii ( fig . 1e ) . within endothelial cells , super resolution microscopy revealed that endogenous vwf and lc3 were clearly separable . nonetheless , at any given time , a substantial fraction of wpbs were closely associated with a lc3 signal ( 37/224 wpbs or 16.7% ) with a smaller percentage of wpbs ( 7/224 or 3.2% ) appearing to be surrounded by a structure consistent with a lc3-coated autophagosome ( fig . 1e and supplemental movie 1 ) . a separate analysis using confocal microscopy came to a similar conclusion , namely that a substantial fraction ( approximately 30% ) of intracellular lc3 and vwf spots had a centroid to centroid distance of less than 1.5 m ( supplemental fig . finally , using two color confocal live cell imaging , we could capture in real time , the apparent direct interaction of a lc3-gfp autophagosome with a vwf - mcherry positive wpb ( fig . nonetheless , additional dual - color ( lc3-mcherry and vwf - gfp ) or single color ( lc3-gfp ) tirf imaging suggested these interactions were rare and that under normal circumstances , autophagosomes do not appear to directly fuse with the plasma membrane ( supplemental movie 2&3 ) . to begin to address whether autophagy modulates vwf biology , we performed lentiviral shrna mediated knockdown of atg7 in human endothelial cells . in atg7 knockdown cells , western blot analysis confirmed a significant reduction in atg7 expression ( fig . assessment of autophagic flux often relies on the simultaneous measurements of the level of expression of the multidomain , signaling adaptor protein p62/a170/sqstm1 , hereafter referred to simply as p62 , along with the ratio of lc3-ii to lc3-i . a rise in p62 levels and a fall in lc3-ii are consistent with impaired autophagic flux . as expected , knockdown of the essential autophagy gene atg7 had the expected effects on these well characterized markers of autophagic flux ( fig . we then asked whether knockdown of atg7 altered the stimulated release of vwf . using an elisa - based method of the culture medium , we detected an increase in vwf secretion following stimulation with histamine or vegf ( fig . we saw similar effects on vwf secretion using an alternative and independent method for knockdown of atg7 that employed sirna ( supplemental fig . the reduction in the stimulated release of vwf was further confirmed by direct western blot analysis of the endothelial cell supernatant ( fig . 2c , d ) . a recent report has suggested that disruption of autophagy might alter intracellular calcium signaling . given the central role of calcium in stimulated exocytosis from the endothelium , we wondered whether such a mechanism might explain our observations . using a calcium sensitive fluorometric probe , we could not however detect significant differences in ligand - induced calcium transients between control and atg7 knockdown cells ( fig . similarly , other proximal signaling events such as extracellular regulated kinase ( erk ) activation appeared to be unaffected by atg7 knockdown ( supplemental fig . 4 ) . we next sought to establish whether other genetic or pharmacological inhibitors of autophagy had similar effects . knockdown of the essential autophagy gene atg5 alone or in combination with atg7 also inhibited vwf release ( fig . following histamine stimulation , the combined knockdown of atg5 and atg7 appeared slightly more effective than single knockdown of either atg7 or atg5 in blocking vwf secretion ( fig . incubation of human endothelial cells with chloroquine or bafilomycin , two known inhibitors of autophagic flux , also resulted in a marked inhibition of ligand - stimulated vwf secretion ( fig . in contrast , knockdown of atg5 or atg7 did not appear to alter ligand - stimulated release of endothelin-1 from endothelial cells ( supplemental fig . 5b ) . in addition , while knockdown of atg6 ( beclin 1 ) was quite effective in reducing atg6 expression ( fig 2i ) , it had only a very modest effect on markers of autophagic flux ( supplemental fig . we next sought to attempt to get a better understanding of why knockdown of an essential autophagy gene such as atg7 might inhibit the secretion of vwf . following synthesis , vwf undergoes a series of processing events in the er and golgi that eventually lead to maturation of the protein and incorporation into wpbs . using a quantitative elisa - based method we determined that the total intracellular level of vwf protein was not significantly different between control and atg7 knockdown endothelial cells ( fig . similar results were observed following knockdown of atg5 or combined atg5 and atg7 knockdown ( supplemental fig . we next sought to analyze whether differences might exist in the processing , maturation and secretion of vwf . the processing of vwf involves a furin - dependent proteoltyic cleavage within the trans - golgi network with the pro - vwf precursor distinguishable from mature vwf based on electrophoretic mobility . we observed that the steady state ratio of pro - vwf to mature vwf was significantly altered in atg7 or atg5 knockdown cell ( fig . consistent with a defect in vwf processing , we also noted a reduction in the number of mature wpbs observed in atg7 knockdown cells ( fig . we saw similar reduction in wpbs in endothelial cells with either atg5 knockdown or following pharmacological inhibition of autophagy using chloroquine or bafilomycin ( supplemental fig . in contrast , knockdown of atg6 did not alter the number of mature wpbs ( supplemental fig . these results suggest that one explanation for our observation with atg5 or atg7 knockdown is that these strategies , while not altering overall vwf expression , appear to inhibit the processing and maturation of vwf into mature wpbs . the formation of wpbs is believed to occur at the trans - golgi network ( tgn ) and autophagy is known to be critical for the homeostatic maintenance of both the er and golgi . furthermore , the ph of wpbs is known to be acidic and this low ph is essential for the shape of the wpbs , as well as vwf tubulation and function . using previously described methods , we were able to establish that wpbs within atg7 knockdown cells exhibited a higher ph than wpbs found in control cells ( fig . 4a and supplemental fig . consistent with previous results , when assessed by confocal microscopy , this higher ph in atg7 knockdown cells resulted in rounder and less rod - shaped wpbs ( fig . this also translated into a statistically significant reduction in the length of wpbs found in atg7 knockdown cells ( fig . similar observations were evident by electron microscopy where we observed that knockdown of atg7 resulted in wpbs that were shorter , rounder and had less distinct tubulation ( fig . 4e ) . to further assess whether in addition to a processing defect , there was also an effect of atg7 on the secretion of mature wpbs , we employed a previously described system using live cell imaging of the secretion of a vwf - gfp chimeric fusion protein . expression of this tagged vwf results in assembly of wpbs containing the co - expressed fluorescently labeled vwf protein ( supplemental fig . consistent with the alterations in processing described above , the number of gfp - positive wpbs was reduced in cells where atg7 was knockdown ( supplemental fig . 9b ) and the wpbs that formed , had an altered , more rounded morphology ( supplemental movies 4 , 5 ) . using this system , in conjunction with total internal reflection fluorescence ( tirf ) microscopy , we could monitor individual secretion events of gfp - positive wpbs that localized near the plasma membrane ( fig . our analysis demonstrated that knockdown of atg7 resulted in reduced wpb secretion after histamine stimulation ( fig . in an effort to extend these observations to an in vivo situation , we conditionally deleted atg7 within endothelial cells by crossing mice harboring atg7 floxed alleles with transgenic mice expressing the previously described ve - cadherin cre recombinase . parental mice both of which bore the genotype atg7 ; ve - cadherin cre were crossed to generate offspring that were either atg7 ; ve - cadherin cre ( hereafter denoted as atg7 ) or atg7 ; ve - cadherin cre ( hereafter denoted as control ) . given the relatively small contribution of endothelial cells to overall organ mass , we did not observe a significant reduction in atg7 expression in the tissues and organs isolated from atg7 mice ( supplemental fig . in contrast , primary cultures of mouse endothelial cells isolated from atg7 mice exhibited the expected reduction in atg7 expression ( fig . the residual expression of atg7 in these cells may represent a combination of incomplete deletion with ve - cadherin cre , as well as potentially some degree of contamination of non - endothelial cells in these primary cultures . nonetheless , this level of atg7 expression was sufficient to induce a reduction in autophagic flux as evidenced by the lc3-i / ii ratio and p62 levels ( fig . similar to our observations employing knockdown of atg7 in human endothelial cells , we did however observe a reduction in the number of mature wpbs in endothelial cells derived from atg7 mice ( fig 5b ) . while conditional deletion of essential autophagy genes in tissues such as brain led to marked alterations in tissue homeostasis and early mortality , atg7 mice exhibited no obvious vascular phenotypes . similarly , atg7 mice exhibited no increase in mortality over the first year of life ( unpublished observations ) . assessment of vascular density demonstrated that atg7 mice were indistinguishable from control animals ( fig . 5c ) . in addition , while autophagy has been implicated in angiogenesis , we saw no consistent or reproducible abnormalities in atg7 mice when post - natal retinal angiogenesis was assessed ( fig . , the endothelium and underlying architecture of large vessels were similar in atg7 and control mice ( fig . we also observed no reduction in the level of vwf expression in atg7 mice either by immunohistochemical ( fig . we next asked whether atg7 mice exhibited defects in vwf secretion . in control mice , injection of epinephrine led to a significant rise in plasma vwf levels ( fig . 6a , fold increase : 1.5 + / 0.04 , n=27 mice , p<0.001 pre versus post ) . this increase in vwf in response to subcutaneous epinephrine injection is similar in magnitude to previous reports . in contrast , atg7 mice failed to respond in a significant fashion to an epinephrine challenge ( fig . 6a , fold increase : 1.17 + / 0.05 , n=13 mice , p = ns pre versus post ) . this lack of stimulated epinephrine release was also evident in mice where atg5 had been conditionally deleted again using ve - cadherin cre . similar to atg7 mice , atg5 mice also failed to respond to epinephrine ( fig . 6a , fold increase : 1.13 + / 0.07 , n=9 mice , p = ns pre versus post ) . neither atg5 mice nor atg7 mice had a significant reduction in basal vwf ( fig . basal levels of circulating p - selectin levels were not altered between control and atg7 mice ( supplemental fig . to therefore further assess the role of autophagy in regulating basal vwf levels , we analyzed mice having a complete , whole body knockout of atg7 . previous studies demonstrated that these total body atg7 knockout mice rarely survive for more than 48 hours after birth . when we analyzed atg7 mice shortly after delivery , we did note a reduction in plasma vwf when compared to wild type littermates , perhaps reflective of a more complete knockout in this model then can be achieved using conditional approaches ( fig . we next sought to further characterize the physiological importance of atg7 in vwf processing in vivo . consistent with the observed in vitro deficit in vwf processing , we noted that on non - reducing gels , plasma vwf analyzed from atg7 mice contained diminished levels of the biologically active , higher molecular weight multimers ( fig . consistent with this reduction in high molecular weight vwf , coupled with the decrement in stimulated vwf release ( fig . 6a ) , we noted a significant increase in measured bleeding time when control mice were compared to atg7 mice ( fig . such differences occurred in the absence of any measurable differences in various hemostatic parameters ( supplemental fig . furthermore , we did not see any obvious abnormalities in platelet morphology or platelet vwf levels that could have potentially contributed to the observed alterations in the measured bleeding time ( supplemental fig . 14 ) . given that one of the many effects for chloroquine is inhibition of autophagy and since this agent is approved for human usage , we next randomized mice to saline or chloroquine ( cq ) injections . after 9 days , basal plasma vwf levels in cq treated and vehicle treated mice were similar ( supplemental fig . nonetheless , direct assessment of hemostasis revealed that similar to atg7 mice , pharmacological inhibition of autophagic flux resulted in a significant prolongation of bleeding time ( fig . 6e and supplemental fig . our data suggests that knockdown or deletion of atg5 or atg7 results in the impaired in vitro and in vivo stimulated secretion of vwf . interestingly , we and others have demonstrated that conditional deletion of atg7 within the pancreatic -cell leads to impaired secretion of insulin . similarly , impaired granular exocytosis is observed following disruption of autophagy within mast cells , the intestinal paneth cell and osteoclasts . thus , our current results , in conjunction with these past observations , suggest that in addition to its known role in intracellular recycling , autophagy plays a fundamental role in the regulated secretion that occurs in certain specialized cell types . it is currently unclear in the case of mast cells , paneth cells or osteoclasts exactly how autophagy regulates granule secretion . our data suggests that autophagosomes and wpbs can directly interact and that vwf protein and wpb remnants appear to be found within autophagosomes . recent evidence suggests that under certain conditions ( e.g. starvation ) , autophagic vacuoles can actually be directly secreted by endothelial cells . consistent with our microscopy observations , proteomic analysis of these secreted endothelial autophagic vacuoles reveals they contain vwf . nonetheless , although our data suggests an interaction between autophagosomes and wpbs , this interaction is relatively rare when assessed by electron microscopy , super resolution microscopy or dual - color tirf imaging . one intriguing possibility is that the observed interaction represents a mechanism to remove damaged or dysfunctional wpbs , in a similar fashion to how mitophagy is thought to remove damaged mitochondria . how autophagosomes could potentially distinguish a normal from a damaged wpb is unclear , although in this regard , the basis of mitophagic selectivity also remains unresolved . finally , it is interesting to note that our observations may have important therapeutic implications . this agent has many effects on cells since it can suppress the acidification of late endosomes , mvbs and lysosomes . chloroquine is widely given for the treatment and prevention of malaria as well as for treatment of certain classes of autoimmune diseases . interestingly , one known side effect of the drug is an increased risk for bleeding and the longer acting derivative hydroxychloroquine has been shown to be effective in humans as a prophylaxis against thromboembolic complications following surgery . our data provides support to the notion that other agents that similarly target autophagic flux might also modulate vwf secretion . as such , targeted pharmacological inhibition of the autophagic pathway may provide a novel approach to reduce thrombus formation .

endothelial secretion of von willebrand factor ( vwf ) from intracellular organelles known as weibel - palade bodies ( wpbs ) is required for platelet adhesion to the injured vessel wall . here , we demonstrate that wpbs are in some cases found near or within autophagosomes and that endothelial autophagosomes contain abundant vwf protein . pharmacological inhibitors of autophagy , or knockdown of the essential autophagy genes atg5 or atg7 , inhibits the in vitro secretion of vwf . furthermore , while mice with an endothelial specific deletion of atg7 have normal vessel architecture and capillary density , these animals exhibit impaired epinephrine - stimulated vwf release , reduced levels of high molecular weight vwf multimers and a corresponding elevation of their bleeding times . endothelial deletion of atg5 or pharmacological inhibition of autophagic flux results in a similar in vivo alteration of hemostasis . thus , autophagy regulates endothelial vwf secretion and transient pharmacological inhibition of autophagic flux may be a useful strategy to prevent thrombotic events .

Introduction Results Proximity of endothelial WPBs to autophagosomes Autophagy regulates Autophagy is required for VWF protein maturation Autophagy regulates in vivo VWF release Discussion Supplementary Material

PMC4872484

mixed reality technologies , including augmented reality ( ar ) and virtual reality ( vr ) , either supplement or completely replace the real world with computer - generated data to facilitate user interaction via one 's natural senses . both vr and ar technologies have been shown to be beneficial in providing contextual learning experiences , enabling the exploration of complex networks of information , and constructing new models of understanding . vr technology , which was all the rage among technophiles in the early 1980s and 1990s , has been utilized in various applications from gaming to engineering . however , early prototypical vr systems came with a hefty price tag . additionally , early vr systems often featured large , cumbersome head - mounted displays ( hmd ) , which were hampered by narrow fields of view ( fov ) , low frame rates , and suboptimal latencies . as a result , the vr experience offered by such systems were not very immersive and rather uncomfortable to wear for extended periods of time . thus , work with vr technology was essentially limited to researchers and developers with access to these proprietary tools . more recently , oculus vr , a company acquired by facebook following a successful kickstarter campaign , is on the verge of releasing the first consumer version of it 's low - cost , lightweight vr platform , the rift . the oculus rift is a vr hmd with a wide fov ( 110 ) , high - definition quality video resolution ( 2160x1200 ) , and a high frame rate ( 90hz ) , ensuring little to no lag with head movement tracking . in addition , the oculus rift relies on an embedded infrared tracking system that enables positional and rotational tracking of the hmd , which has implications for clinical research . putting on the headset replaces an individual 's field of vision with a digital image , where each eye looks through a different lens to create a stereoscopic three - dimensional ( 3d ) effect . a few studies have investigated the role of vr and ar technologies as tools for medical education and training , where rich contextual learning environments are required . much of this work stems back to the early 2000s , when these technologies were still in their infancy . a handful of studies have purported investigating the role of vr technology in pathology ; however , there are practically no investigations that leverage the current iterations of vr or ar technology . several pathology laboratories are currently in the process of transitioning to using digital imaging systems , such as whole slide imaging ( wsi ) . wsi platforms emulate traditional light microscopy by digitizing glass slides using specialized hardware ( e.g. , whole slide scanners ) and software ( e.g. , vendor - specific or vendor - agnostic digital slide viewers ) . digital slides obtained from wsi platforms are typically viewed in two dimensions either using a desktop personal computer monitor / display or mobile device ( e.g. , tablet ) . current complaints from users of wsi are that navigation of digital slides is slow and tedious , especially with input devices such as a computer mouse . indeed , employing gaming controllers such as those used by sony playstation to create an ultra - high - speed wsi viewing system appear to improve user viewing satisfaction . pathologists also complain that when viewing a digital slide on a computer monitor , they lose the benefit of peripheral vision they are used to when looking at a glass slide with a conventional light microscope . this may partly explain why pathologists like using very large displays made up of many monitors ( e.g. , power wall ) to interact with virtual slides . to the best of our knowledge , we are not aware of any studies regarding regarding digital pathology slides being viewed in a vr environment . therefore , we sought to explore the use of the oculus rift for examining digital pathology slides . an oculus rift development kit 2 ( dk2 ) was connected through usb cables to a 64-bit computer ( hp z440 workstation , intel xeon cpu e5 - 1650 v3 , 32 gb ddr4 - 2133 , nvidia geforce gtx titan x gpu , 512 gb pcie ssd , 1 tb 7200rpm hdd ) running microsoft windows 10 ( 64-bit ) . whole slide images acquired for this study were stored on a remote server and viewed using imagescope ( leica biosystems , wetzlar , germany ) software . additional software was necessary to display two - dimensional ( 2d ) whole slide images in a stereoscopic 3d virtual environment . initially , we investigated the possibility of creating our own vr software using the development kit provided by oculus ( sdk 0.8.0.0 ) and a unity 3d plugin ( http://www.unity3d.com/unity ) , a popular cross - platform game engine . a base example was built in the form of an empty , cubic room with static images used to wallpaper the walls [ figure 1 ] . for ease of use , we opted to instead use commercially available virtual desktop software ( v0.9.34 ) ( http://www.vrdesktop.net/ ) for navigation of digital slides . navigation of digital whole slides with the oculus rift was tested using keyboard controls , an optical mouse , and a leap motion controller ( https://www.leapmotion.com/product/vr ) . the leap motion controller relied on two monochromatic infrared cameras and three infrared light - emitting diodes to generate 3d positional data . leap motion enabled users to perform hand gesture - controlled navigation of digital slides in a vr environment . example of a stereo rendering constructed using the oculus sdk and unity three - dimensional plugin de - identified glass slides from twenty cases ( n = 20 ) were obtained from a lymph node teaching set . cases were randomly selected by two pathologists who were nonparticipants in the viewing phase of the study . ten of the twenty cases harbored malignant diagnoses including metastatic ductal carcinoma , lobular carcinoma , signet ring cell carcinoma , leiomyosarcoma , papillary thyroid carcinoma , malignant melanoma , squamous cell carcinoma , prostatic adenocarcinoma , endometrioid endometrial adenocarcinoma , and hodgkin 's lymphoma . the remaining ten cases harbored benign diagnoses including reactive paracortical lymphoid expansion , normal lymph node , fatty infiltration of an inguinal and pelvic lymph node , tuberculous lymphadenitis , epstein barr virus - associated lymphadenitis , cat scratch disease , hiv - associated lymphadenitis , sarcoidosis , and sinus histiocytosis . a single glass slide from each of the twenty cases was digitized using an aperio scanscope xt wsi scanner ( leica biosystems , wetzlar , germany ) with an olympus uplansapo 20x/0.75 objective . the resultant whole slide images were acquired as jpeg2000 encoded svs files and were not further compressed . digital slides were first viewed using the oculus rift dk2 and virtual desktop application . after a washout period of 2 weeks , the participants again evaluated the same digital slides on a 27-inch in - plane switching ( ips ) 5k display ( hp zr27q ) with a resolution of 4096 2160 pixels , or a 24-inch ips display ( hp zr2440w ) with a resolution of 1920 200 , both with a landscape orientation . three , board - certified , attending pathologists with subspecialty expertise in cytopathology , breast / gynecologic , and gastrointestinal pathology were recruited for review of the aforementioned digital slides . each of the three participants had prior experience in viewing digital pathology slides using traditional wsi modalities . the pathologists were first trained to navigate whole slide images in our vr environment [ figure 2 ] . the primary endpoints assessed included diagnostic concordance and time to read ( interpret ) digital slides using both modalities . diagnostic concordance was defined as ( 1 ) accurate classification of cases as either benign or malignant and ( 2 ) rendering a specific pathologic diagnosis ( i.e. , metastatic carcinoma ) . the pathologists were also asked to rate image quality ( on a scale of 110 ) , ease of navigation ( on a scale of 110 ) , and their diagnostic confidence ( on a scale of 110 ) for both modalities . all primary and secondary endpoints were collected on a per slide / case basis , and the average values for each pathologist were tabulated as overall " values . virtual reality - based digital slide navigation using oculus rift development kit 2 , virtual desktop , and a web - based whole slide imaging browser an oculus rift development kit 2 ( dk2 ) was connected through usb cables to a 64-bit computer ( hp z440 workstation , intel xeon cpu e5 - 1650 v3 , 32 gb ddr4 - 2133 , nvidia geforce gtx titan x gpu , 512 gb pcie ssd , 1 tb 7200rpm hdd ) running microsoft windows 10 ( 64-bit ) . whole slide images acquired for this study were stored on a remote server and viewed using imagescope ( leica biosystems , wetzlar , germany ) software . additional software was necessary to display two - dimensional ( 2d ) whole slide images in a stereoscopic 3d virtual environment . initially , we investigated the possibility of creating our own vr software using the development kit provided by oculus ( sdk 0.8.0.0 ) and a unity 3d plugin ( http://www.unity3d.com/unity ) , a popular cross - platform game engine . a base example was built in the form of an empty , cubic room with static images used to wallpaper the walls [ figure 1 ] . for ease of use , we opted to instead use commercially available virtual desktop software ( v0.9.34 ) ( http://www.vrdesktop.net/ ) for navigation of digital slides . navigation of digital whole slides with the oculus rift was tested using keyboard controls , an optical mouse , and a leap motion controller ( https://www.leapmotion.com/product/vr ) . the leap motion controller relied on two monochromatic infrared cameras and three infrared light - emitting diodes to generate 3d positional data . leap motion enabled users to perform hand gesture - controlled navigation of digital slides in a vr environment . example of a stereo rendering constructed using the oculus sdk and unity three - dimensional plugin de - identified glass slides from twenty cases ( n = 20 ) were obtained from a lymph node teaching set . cases were randomly selected by two pathologists who were nonparticipants in the viewing phase of the study . ten of the twenty cases harbored malignant diagnoses including metastatic ductal carcinoma , lobular carcinoma , signet ring cell carcinoma , leiomyosarcoma , papillary thyroid carcinoma , malignant melanoma , squamous cell carcinoma , prostatic adenocarcinoma , endometrioid endometrial adenocarcinoma , and hodgkin 's lymphoma . the remaining ten cases harbored benign diagnoses including reactive paracortical lymphoid expansion , normal lymph node , fatty infiltration of an inguinal and pelvic lymph node , tuberculous lymphadenitis , epstein barr virus - associated lymphadenitis , cat scratch disease , hiv - associated lymphadenitis , sarcoidosis , and sinus histiocytosis . a single glass slide from each of the twenty cases was digitized using an aperio scanscope xt wsi scanner ( leica biosystems , wetzlar , germany ) with an olympus uplansapo 20x/0.75 objective . the resultant whole slide images were acquired as jpeg2000 encoded svs files and were not further compressed . after a washout period of 2 weeks , the participants again evaluated the same digital slides on a 27-inch in - plane switching ( ips ) 5k display ( hp zr27q ) with a resolution of 4096 2160 pixels , or a 24-inch ips display ( hp zr2440w ) with a resolution of 1920 200 , both with a landscape orientation . three , board - certified , attending pathologists with subspecialty expertise in cytopathology , breast / gynecologic , and gastrointestinal pathology were recruited for review of the aforementioned digital slides . each of the three participants had prior experience in viewing digital pathology slides using traditional wsi modalities . the pathologists were first trained to navigate whole slide images in our vr environment [ figure 2 ] . the primary endpoints assessed included diagnostic concordance and time to read ( interpret ) digital slides using both modalities . diagnostic concordance was defined as ( 1 ) accurate classification of cases as either benign or malignant and ( 2 ) rendering a specific pathologic diagnosis ( i.e. , metastatic carcinoma ) . the pathologists were also asked to rate image quality ( on a scale of 110 ) , ease of navigation ( on a scale of 110 ) , and their diagnostic confidence ( on a scale of 110 ) for both modalities . all primary and secondary endpoints were collected on a per slide / case basis , and the average values for each pathologist were tabulated as overall " values . virtual reality - based digital slide navigation using oculus rift development kit 2 , virtual desktop , and a web - based whole slide imaging browser technically , digital images were able to be viewed in a vr environment wearing the oculus rift dk2 . all input methods to navigate slides worked satisfactorily . however , the leap motion method resulted in the most fatigue after prolonged hand gestures . all three reviewers confirmed that digital pathology slides were easily viewable in a vr environment using the oculus rift dk2 . there was 90% diagnostic concordance between the traditional method of reviewing wsi on a flat computer monitor and the vr method . the variation in time to interpret digital pathology slides did reach statistical significance when tested using an unpaired t - test ( two - tailed p = 0.0009 ) . variations in image quality and ease of navigation also reached statistical significance when tested using unpaired t - tests ( two - tailed p = 0.0001 and 0.027 , respectively ) . the variations in diagnostic confidence , however , did not reach statistical significance ( two - tailed p = 0.15 ) . there were no significant side effects ( e.g. , motion sickness ) associated with the use of the oculus rift ; however , the participants did note that the vr headset did feel heavy after prolonged usage . this proof of concept study represents the first attempt to examine pathology whole slide images in a vr environment . it is important to note , however , that the aim of this initial study was on proof - of - concept and feasibility as opposed to a comprehensive vr validation study . as such , the small sample size of only twenty cases likely limited our ability to discern meaningful differences between vr and traditional modalities . nonetheless , our findings demonstrate that digital pathology slides can be successfully interpreted using vr . vr technology today has become readily accessible so that it no longer requires the use of very expensive , complicated , or proprietary hardware or software . therefore , pathology departments currently using wsi can readily leverage vr technologies , such as the oculus rift , without significant investment . a widely available , commercial version of the oculus rift is expected in 2016 . in order for digital slides to be viewed with the oculus rift dk2 , we had to integrate a wsi viewer into a virtual environment , map the wsi viewer navigation controls into this environment , and utilize the virtual environment to create a novel 3d interaction pattern for navigating digital slides . navigation was possible using standard input devices ( e.g. , keyboard , mouse ) and natural senses ( e.g. , hand gestures ) . we utilized the well - known aspects of vr classification scheme to determine which software to employ in this study . this classification includes six major factors of vr : interaction , immersion , simulation , visualization , real space , and autonomous agents . to create a truly immersive and engaging vr experience a high frame rate and low hmd latency are perhaps the most essential factors in the creation of a truly immersive vr experience . the frame rate is the frequency at which a device displays continuous images for human visual systems to perceive . while the majority of current media is run at 3060 frames per second , vr media requires higher frame rates to ensure smooth movements and prevent motion sickness . the dk2 makes virtual objects feel almost tangible by supposedly maintaining a frame rate of up to 75 frames per second . unfortunately , the frame rate was varied significantly as users navigated the digital slides , resulting a noticeable judder . latency , in the context of vr , refers to the total time between movement of the user 's head and the updated image being displayed on the screen . thus , a lower latency results in a more immersive and comfortable user experience . low latency head tracking is part of what sets the rift apart from other vr technologies . first , the image quality on the oculus rift dk2 was limited by the resolution of the organic light - emitting diode display panel in the headset , which only has a resolution of 960 1080 per eye . in addition , there are hardly any medical applications available for use with vr . while the vr environment we developed provides pathologists with a wide fov because the digital images themselves were not acquired in 3d , the vr environment only created a virtual projection of the 2d whole slide image floating in a simulated 360 environment . this may explain why the oculus rift did not significantly enhance the navigation of digital slides . moreover , navigation may have been negatively impacted by the input devices ( mouse and keyboard ) and pathologists were confined to use for the comparative study . oculus is currently developing a pair of lightweight , wireless , handheld motion controllers that may perform better than traditional input devices . in addition , our participants did not experience any serious negative side effects largely because they did not spend much time immersed in the vr environment . given the well - documented side effects associated with vr immersion ( e.g. , nausea , eye - strain , and seizures ) , the long - term usage of vr in clinical practice is an area that deserves further investigation . unfortunately , many pathologists have encountered frustration when attempting to review digital pathology slides because of navigation difficulties encountered with certain input devices or viewing limitations with mobile computing devices such as laptops . hence , the availability of an immersive , private , and more natural means of digital slide review would be very helpful . a vr headset such as oculus rift could allow pathologists to more easily perform telepathology consultations in a secure environment . vr also has a great potential use in pathology teaching , especially if combined with 3d images ( e.g. , 3d models of gross pathology specimens ) . our group is investigating the methods for generating 3d digital images of tissue blocks that may be benefited from stereoscopic 3d visualization in a vr environment .

background : digital slides obtained from whole slide imaging ( wsi ) platforms are typically viewed in two dimensions using desktop personal computer monitors or more recently on mobile devices . to the best of our knowledge , we are not aware of any studies viewing digital pathology slides in a virtual reality ( vr ) environment . vr technology enables users to be artificially immersed in and interact with a computer - simulated world . oculus rift is among the world 's first consumer - targeted vr headsets , intended primarily for enhanced gaming . our aim was to explore the use of the oculus rift for examining digital pathology slides in a vr environment.methods:an oculus rift development kit 2 ( dk2 ) was connected to a 64-bit computer running virtual desktop software . glass slides from twenty randomly selected lymph node cases ( ten with benign and ten malignant diagnoses ) were digitized using a wsi scanner . three pathologists reviewed these digital slides on a 27-inch 5k display and with the oculus rift after a 2-week washout period . recorded endpoints included concordance of final diagnoses and time required to examine slides . the pathologists also rated their ease of navigation , image quality , and diagnostic confidence for both modalities.results:there was 90% diagnostic concordance when reviewing wsi using a 5k display and oculus rift . the time required to examine digital pathology slides on the 5k display averaged 39 s ( range 10120 s ) , compared to 62 s with the oculus rift ( range 15270 s ) . all pathologists confirmed that digital pathology slides were easily viewable in a vr environment . the ratings for image quality and diagnostic confidence were higher when using the 5k display.conclusion:using the oculus rift dk2 to view and navigate pathology whole slide images in a virtual environment is feasible for diagnostic purposes . however , image resolution using the oculus rift device was limited . interactive vr technologies such as the oculus rift are novel tools that may be of use in digital pathology .

INTRODUCTION METHODS Virtual Reality Setup Study Design RESULTS CONCLUSION Financial Support and Sponsorship Conflicts of Interest

PMC4135198

with the rapid increase in the proportion of elderly in japan , the proportion of adult diseases such as cancer , ischemic heart disease , cerebrovascular disease , and diabetes mellitus has also increased , now accounting for about 60% of the causes of mortality1 , 2 . exercise plays a crucial role in the prevention of adult disease and is recommended to maintain or improve health3 , 4 . it has been reported that a certain intensity of exercise is required for improvements in health and physical strength5,6,7 . at least a moderate exercise intensity is required to improve the health of adults6 , but comorbidities in the elderly may restrict their ability to exercise . therefore , exercise should be prescribed at a suitable intensity for the elderly , i.e. , the intensity at which the benefits of exercise can be gained but risks of exercise avoided . the balance between benefit and safety varies with the physical condition of the individual but is considered to occur at a lower intensity in elderly people with existing disease than in healthy young adults . therefore , exercise intensity in the elderly must be carefully adjusted . anaerobic threshold ( at ) has been proposed as an objective criterion for precise adjustment of exercise intensity8 . at is the exercise intensity just after which energy production by anaerobic metabolism is added to aerobic metabolic energy production during incremental exercise . it is estimated by the lactate threshold ( lt ) , which is the workload at which the blood lactate concentration abruptly increases , and by the ventilatory threshold ( vt ) , which is the workload at which the rate of pulmonary minute ventilation abruptly increases nonlinearly during a progressive exercise test9 , 10 . it has been reported that both thresholds are safe and effective criteria11 , 12 . however , both thresholds have disadvantages ; specifically , the gas analyzer used to measure expired gas for determination of vt is expensive and is limited to use in a laboratory setting , and blood collection for determination of lt is invasive . one is the double - product breakpoint ( dpbp)13 , or the point at which double product ( dp ) , the product of heart rate and systolic blood pressure , abruptly increases during a progressive exercise test . the dpbp can be calculated from the intersection of two regression lines , and its validity has already been established based on the high correlation between dpbp and at ( r = 0.87 , p < 0.001)14,15,16 . another method for estimating at is heart rate threshold ( hrt ) , which uses only heart rate17 , 18 . hrt is obtained from the relationship between running speed and heart rate . the running speed heart rate relationship is linear from low to submaximal speeds and curvilinear from submaximal to maximal speeds . the speed of transition from the linear to the curvilinear phase coincides with the beginning of a sharp accumulation of blood lactate18 . however , it has been reported that the dpbp method is inferior to expired gas analysis in detecting at14 and that the hrt occurs at a higher workload than at19 . for this reason , it is necessary to develop a more sensitive indicator or to create a method combining a number of indicators to improve the ability to detect at and thus prescribe suitable exercise to subjects with reduced exercise tolerance . both systolic blood pressure and heart rate , the components of dp , are controlled by the sympathetic nervous system . the plasma catecholamine concentration , which reflects sympathetic nervous system activity during incremental exercise , has been shown to start to rise abruptly at a workload similar to the lt . in addition , it has been reported that the plasma catecholamine concentration correlates with the blood lactate concentration20 , 21 . this suggests that dp may reflect changes in sympathetic nervous system activity and blood lactate concentration through the plasma catecholamine concentration . therefore , it is possible to detect the at more precisely if an index is used that is a more sensitive reflection of sympathetic nervous system activity . the proposed index of sympathetic nervous system activity is salivary -amylase , an enzyme in saliva that has been reported to be controlled by the sympathetic - adrenal - medullary system22 , 23 and to be a sensitive indicator of the activity of the sympathetic nervous system24 . therefore , it is possible to detect changes in blood lactate concentration indirectly by measuring salivary -amylase , a measurement that can be performed quickly under test conditions . investigated the relationship between the salivary threshold ( tsa ) , the workload at which salivary -amylase abruptly increases , and the lt by measuring salivary -amylase and blood lactate concentrations during a multistage incremental exercise25 . the authors concluded that tsa had a high correlation with the lt ( r = 0.95 , p < 0.001 ) , and the detection rate was 80% . the results suggested that the high correlation between the tsa and lt , as well as the ease of measuring salivary -amylase , makes tsa a valid and useful estimate of at . moreover , no report has investigated the relationship between tsa and other methods of estimating at . determination of whether the detection rate is improved by combining tsa and dpbp would provide meaningful information for exercise prescription , particularly for elderly people with preexisting disease . the purpose of this study was to assess the validity of salivary -amylase as a method for estimating at quickly and to clarify the relationship between salivary -amylase and dpbp in order to establish a way to adjust exercise intensity to a safe and effective range . the subjects of this study were 11 healthy young adults ( 23.8 1.8 years , height 173 5.0 cm , body weight 64.6 6.3 kg , body mass index 21.7 1.3 none of the subjects reported any neurological or vestibular disorders , orthopedic conditions , or oral cavity diseases before participating in this study . we used an electrically braked cycle ergometer ( aerobike 75xl , combi co. ltd . , the timing of the experiment ( 6:00 p.m. to 8:00 p.m. ) was chosen to minimize the influence of circadian variation in salivary -amylase26 . since the activity of salivary -amylase can be influenced by alcohol , medications , food , and caffeine , all subjects were instructed not to drink any alcohol the day before measurement and not to consume food or caffeine 2 hours before measurement . after a rest period of 3 min on the ergometer , subjects were instructed to try to maintain a cadence of 50 rpm while the workload level was increased by 20 watts per 3 min beginning with 10 watts . the exercise test was continued until subjects could no longer maintain the prescribed cadence due to fatigue or until completion of 2 stages following that at which at was observed . oxygen consumption ( vo2 ) , carbon dioxide production ( vco2 ) , and ventilatory equivalent ( ve ) were measured using a breath - by - breath gas analyzer ( ae-300 , minato medical science , osaka , japan ) . systolic blood pressure and heart rate were measured during the last 30 seconds of each stage of the incremental exercise test . heart rate was measured using an electrocardiogram monitor ( bsm-2401 , nihon kohden , tokyo , japan ) . the dpbp was determined using a computer algorithm as follows : the linear regression lines of dp as a function of workload were calculated for all possible divisions of the data . dpbp was determined by choosing the intersection , or breakpoint , of the 2 lines representing the minimum residual sum of squares from among various intersections of the 2 lines . salivary -amylase was measured using a portable salivary amylase analyzer ( salivary amylase monitor , nipro , osaka , japan ) . it was verified that within the analyzer s linear range ( 10230 ku / l ) , this handheld monitor s accuracy ( r = 0.989 ) , precision ( coefficient of variation < 9% ) , and measurement repeatability ( range 3.1% to + 3.1% ) approached those of a more elaborate laboratory - based automated clinical chemistry analyzer ( olympus america inc . , center valley , pa , usa ) . salivary -amylase was measured from saliva samples collected during the last 30 seconds of each stage of an incremental exercise test by inserting the reagent test strip directly into the subject s oral cavity . the mask for expired gas analysis was briefly removed to obtain the saliva sample and then replaced after saliva collection . workload at the at , tsa , and dpbp were compared using one - way anova to investigate the difference in workload between each index . pearson s correlation coefficients were used to assess the relationship between workload at the at and tsa and between the at and dpbp . in addition , with tsa-1 ( or dpbp-1 ) representing 1 workload prior to that corresponding to tsa ( or dpbp ) , tsa-2 representing 2 workloads prior , tsa-3 representing 3 workloads prior , and so on , tsa , tsa-1 , tsa-2 , and tsa-3 and workloads for dpbp , dpbp-1 , dpbp-2 , and dpbp-3 were compared by one - way anova with repeated measures to identify the dynamics of salivary -amylase and dpbp associated with an incrementally increasing workload . subject characteristicsage ( yr)23.8 1.8height ( m)1.73 0.05weight ( kg)64.6 6.3bmi ( kg / m)21.7 1.3hr at rest ( beats / min)70.0 11.4hr at at ( beats / min)116.2 16.4vo2 at at ( ml / min)1,170.2 189.3vo2/w at at ( ml / minkg)18.1 2.3salivary -amylase at rest ( ku / l)22.2 27.5salivary -amylase at tsa ( ku / l)40.0 18.1dp at rest ( mmhg beats / min)8,740.4 1,994.7dp at dpbp ( mmhg beats / min)14,868.2 3,153.1data presented as mean sd . bmi , body mass index ; at , anaerobic threshold ; dp , double product ; dpbp , double - product breakpoint ; hr , heart rate ; tsa , salivary threshold ; w , watt . in the present study , at could be determined in all subjects ( 100% ) . however , tsa and dpbp could not be detected in 2 of the subjects ( detection rate of 82% for both methods ) because the increase was not abrupt enough to identify 2 regression lines clearly . in the following analysis , therefore , data for the subjects whose tsa and dpbp could not be detected were excluded . the results of one - way anova revealed that there was no significant difference among workloads at the at , dpbp , and tsa ( table 2table 2 . power output at at , tsa , and dpbpat ( w)70.0 28.3tsa ( w)61.1 28.5dpbp ( w)65.6 21.9at , anaerobic threshold ; dpbp , double - product breakpoint ; tsa , salivary threshold ; w , watt ) . bmi , body mass index ; at , anaerobic threshold ; dp , double product ; dpbp , double - product breakpoint ; hr , heart rate ; tsa , salivary threshold ; w , watt at , anaerobic threshold ; dpbp , double - product breakpoint ; tsa , salivary threshold ; w , watt there were significant correlations between at and tsa ( r = 0.951 , p < 0.01 ) and between at and dpbp ( r = 0.940 , p < 0.01 ) . the results of one - way anova with repeated measures identified the significant main effects of salivary -amylase ( f1.58 , 12.6 = 24.0 , p < 0.01 , partial = 0.75 ) and dp ( f1.34 , 10.3 = 44.7 , p < 0.01 , partial = 0.85 ) , and post hoc analysis using bonferroni s test was executed . the results showed that there were significant differences between tsa and tsa-1 ( p < 0.05 ) , between tsa and tsa-2 ( p < 0.01 ) , and between tsa and tsa-3 ( p < 0.01 ) . however , there were no significant differences between tsa-1 and tsa-2 , between tsa-1 and tsa-3 , or between tsa-2 and tsa-3 . the purpose of this study was to assess the validity of tsa as a workload index by clarifying the relationship between at , which was measured by analysis of expired gas , and tsa , which represented the workload at which salivary -amylase abruptly increased . we also investigated the relationship between dpbp , which has been established as a method for quickly estimating at , and tsa in order to assess the usability of tsa as another method for quickly estimating at . the results of this study indicate significant correlations between at and tsa ( r = 0.951 , p < 0.01 ) . furthermore , a significant difference was not observed in the workload at the at and tsa . this result suggests that tsa has validity as a method for estimating at quickly , which confirms results reported by calvo et al25 . yamamoto et al . investigated the activity of the parasympathetic and sympathetic nervous systems during exercise using heart rate variability . the authors revealed that the indicator of parasympathetic nervous system activity decreased progressively from rest to a workload equivalent to 60% vt and that the indicator of sympathetic nervous system activity increased only when exercise intensity exceeded vt28 . these results suggest that the activity of the sympathetic nervous system changed at the vt , which was equivalent to the at , and that at might be estimated by monitoring the activity of the sympathetic nervous system . moreover , it has been reported that the salivary -amylase concentration increases with increased physical activity , such as treadmill exercise29 , running30 , 31 , and cycle exercise24 , 32 . . investigated salivary -amylase during walking , jogging , and running and reported that jogging and running increased salivary -amylase but that walking did not affect salivary -amylase24 . this result confirmed that the increase in salivary -amylase was caused by physical activity exceeding a certain workload ( i.e. , the at ) . chatterton et al . also investigated the correlation between salivary -amylase and plasma catecholamines and showed significant correlations between them ( r = 0.64 for norepinephrine and r = 0.49 for epinephrine)24 . this result directly indicates that salivary -amylase reflects the activity of sympathetic nervous system . since salivary -amylase is an index that sensitively reflects the activity of the sympathetic nervous system and the activity of the sympathetic nervous system is caused by workloads above the at , it is suggested that tsa , which is the point of inflection of salivary -amylase , is a good index by which to estimate at . the results of the present study also indicated that tsa was at least as good as dpbp as a method for estimating at quickly because the correlation between the at and tsa was 0.951 ( p < this result suggests that salivary -amylase reflects the activity of the sympathetic nervous system more sensitively than dp , the components of which are controlled by the sympathetic nervous system . spence et al . compared the systolic and diastolic blood pressure responses to exercise before the at with those after the at by using a cycle ergometry33 . the authors concluded that the rate of increase of systolic blood pressure significantly increased after the at . tanaka et al . also demonstrated that the slope of a regression line of systolic blood pressure and heart rate increased after the at by comparing the slope of a regression line of systolic blood pressure and heart rate before and after the at13 . however , other researchers reported that the slope of a regression line of systolic blood pressure and heart rate may not increase after the at . riley et al . investigated the relationship between dpbp and at , as determined from analysis of expiration gas , by measuring systolic blood pressure and heart rate during an exercise test14 . their results indicated that , although dpbp and at are strongly correlated , the rates of increase of systolic blood pressure and heart rate do not always increase after the at . in the study of riley et al . , the rate of increase of systolic blood pressure increased after the at for some subjects but that for heart rate did not ; other subjects indicated the opposite . furthermore , conconi et al . researched the relationship between hrt and lt by obtaining the hrt , which was calculated from running speed and heart rate18 . the results indicated that there was a strong correlation between hrt and lt and that increased running speed involved an increase in heart rate . however , the rate of increase of heart rate after the hrt decreased , which is in contrast to the results of tanaka13 . both riley et al.14 and conconi et al.18 demonstrated that changes in systolic blood pressure and heart rate before and after the at differed among individuals . dp is highly correlated with at because it consists of 2 variables , so that even if only 1 variable shows unique changes , the other variable is able to change in a complementary manner . however , using the product of variables that vary among individuals may lead inaccurate estimation of at . salivary -amylase is therefore superior to dp in the degree of correlation to at because salivary -amylase can measure the activity of the sympathetic nervous system using only 1 variable . there are other ways in which tsa is superior to dpbp for estimation of at . to calculate dp , blood pressure and heart rate must be measured , but blood pressure measurement temporarily restricts subjects physical activity . additionally , measurement of heart rate requires equipment such as an electrocardiograph monitor , thus restricting the measurement environment . in contrast , measurement of salivary -amylase requires only insertion of the reagent test strip directly into the subject s oral cavity to collect saliva samples without the need to interrupt activity , and it can be performed in various environments . thus , salivary -amylase estimates at simply and has the advantages of easier measurement and more sensitive reflection of sympathetic nervous system activity than can be achieved with dp . interestingly , the results of the present study suggest that a more correct estimation of at can be achieved by combining tsa and dpbp . in this research , although at was identified in all subjects using expired - gas analysis , tsa and dpbp were unable to be determined in 2 subjects . however , because data allowing determination of dpbp were always collected , combining tsa and dpbp allowed identification of at in all subjects . this finding suggests that the variables salivary -amylase , systolic blood pressure , and heart rate are controlled by the sympathetic nervous system but that the expression of these variables in vivo differs among individuals . therefore , inclusion of all the variables in the estimation of at may allow a more precise estimation . at estimation using salivary -amylase is useful for subjects in whom tsa can be identified even if this is the only method used because tsa and at are strongly correlated . however , the combination of dpbp and tsa might be desirable for some subjects , since tsa was unable to be identified in about 18% of our study subjects . while it is better to measure salivary -amylase to estimate at because of its superiority to dpbp in the degree of correlation with at , if tsa is not able to be determined , dp can be calculated to complement the detection of at . as a result , at can be determined with a detection rate as high as that for expired - gas analysis . this result is in line with the study of calvo et al.25 , who mentioned that differences among subjects in their hydration state before reporting to the laboratory or in the contribution of each salivary gland to the production of total saliva explained the inability to detect tsa in 20% of their subjects . in this study , although we encouraged subjects to drink water before measurement and allocated a point under the tongue for saliva collection in order to minimize these influences , the detection rate was not improved . further studies are needed to improve the precision of measurement of salivary -amylase itself . the results of this study clarified that tsa is a useful index for estimating at during an incrementally increasing workload . tsa was not able to be detected in 2 subjects , but in such cases , the rate of determination of at can be improved by combining tsa with dpbp . determination of at using salivary -amylase is superior to other methods because the measurement device is easily transportable , making measurement possible in various environments , and because of the noninvasiveness of the technique . we believe that the ability to adjust exercise to the optimal intensity in various environments using salivary -amylase can make exercise safer and more effective .

[ purpose ] the purpose of this study was to clarify the validity of salivary -amylase as a method of quickly estimating anaerobic threshold and to establish the relationship between salivary -amylase and double - product breakpoint in order to create a way to adjust exercise intensity to a safe and effective range . [ subjects and methods ] eleven healthy young adults performed an incremental exercise test using a cycle ergometer . during the incremental exercise test , oxygen consumption , carbon dioxide production , and ventilatory equivalent were measured using a breath - by - breath gas analyzer . systolic blood pressure and heart rate were measured to calculate the double product , from which double - product breakpoint was determined . salivary -amylase was measured to calculate the salivary threshold . [ results ] one - way anova revealed no significant differences among workloads at the anaerobic threshold , double - product breakpoint , and salivary threshold . significant correlations were found between anaerobic threshold and salivary threshold and between anaerobic threshold and double - product breakpoint . [ conclusion ] as a method for estimating anaerobic threshold , salivary threshold was as good as or better than determination of double - product breakpoint because the correlation between anaerobic threshold and salivary threshold was higher than the correlation between anaerobic threshold and double - product breakpoint . therefore , salivary threshold is a useful index of anaerobic threshold during an incremental workload .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC5255528

the first direct detection of gravitational waves ( gws ) , widely expected in the mid 2010s with advanced ground - based interferometers [ 219 , 2 ] , will represent the culmination of a fifty - year experimental quest . soon thereafter , newly plentiful gw observations will begin to shed light on the structure , populations , and astrophysics of mostly dark , highly relativistic objects such as black holes and neutron stars . in the low - frequency band that will be targeted by space - based detectors ( roughly 10 to 1 hz ) , gw observations will provide a census of the massive black - hole binaries at the center of galaxies , and characterize their merger histories ; probe the galactic population of binaries that include highly evolved , degenerate stars ; study the stellar - mass objects that spiral into the central black holes in galactic nuclei ; and possibly detect stochastic gw backgrounds from the dynamical evolution of the very early universe . thus , there are very strong astrophysical motivations to observe the universe in gws , especially because the systems and phenomena that can be observed in this fashion are largely orthogonal to those accessible to traditional electromagnetic ( em ) and astroparticle astronomy . einstein s theory of gravity , general relativity ( gr ) , has been confirmed by extensive experimental tests ; but these have largely been confined to the solar system , where gravity is well approximated by newtonian gravity with small corrections . a few tests , based on observations of binary compact - object systems , have confirmed the weakest ( leading - order ) effects of gw generation . by contrast , observation of strong gws will provide the first direct observational probe of the dynamical , strong - field regime of gr , where the nature and behavior of gravity can be significantly different from the newtonian picture . gws are prima facie the perfect probe to investigate gravitation , since they originate directly from the bulk motion of gravitating matter , relieving the need to understand and model the physics of other intermediate messengers , typically photons from stellar surfaces or black - hole surroundings . already today we can rely on a very sophisticated understanding of the analytical and numerical techniques required to model gw sources and their gw emission , including the post - newtonian expansion [ 84 , 190 ] , black - hole perturbation theory , numerical relativity for vacuum spacetimes , spacetimes with gases or magnetized plasmas , and much more . that these techniques should have been developed so much in the absence of a dialogue with experimental data ( except for the binary pulsar ) is witness to the great perceived promise of gw astronomy . for a birds - eye view of the field , see the living review by sathyaprakash and schutz , who cover the physics of gws , the principles of operation of gw detectors , the nature of gw sources , the data analysis of gw signals , and the science payoffs of gw observations for physics , astrophysics , and cosmology . this review focuses on the opportunities to challenge or confirm our understanding of gravitational physics that will be offered by forthcoming space - based missions to observe gws in the low - frequency band between 10 and 1 hz . most of the literature on this subject has focused on one mission design , lisa ( the laser interferometer space antenna [ 64 , 252 , 370 ] ) , which was studied jointly by nasa and esa between 2001 and 2011 . in 2011 , budgetary and programmatic reasons led the two space agencies to end this partnership , and to pursue space - based gw detection separately , studying cheaper , rescoped lisa - like missions . esa s proposed elisa / ngo would be smaller than lisa , fly on orbits closer to earth , and operate interferometric links only along two arms . in 2012 elisa was considered for implementation as esa s first large mission ( l1 ) in the cosmic vision program . a planetary mission was selected instead , but elisa will be in the running for the next launch slot ( l2 ) , with a decision coming as soon as 2014 . nasa ran studies on a broader range of missions , including several variants of lisa to be implemented by nasa alone , as well as options with a geocentric orbit ( omega ) , and without drag - free control ( lagrange ) . the final study report concludes that scientifically compelling missions can be carried out for less , but not substantially less , than the full lisa cost ; that scientific performance decreases far more rapidly than cost ; and that no design choice or technology can make a dramatic reduction in cost without much greater risks . the nasa study noted the possibility of participation in the esa - led elisa mission ( if selected by esa ) as a minority partner . whatever specific design is eventually selected , it is likely that its architecture , technology , and scientific reach will bear a strong resemblance to lisa s ( with the appropriate scalings in sensitivity , mission duration , and so on ) . thus , the research reviewed in this article , which was targeted in large part to lisa , is still broadly relevant to future missions . such lisa - like observatories are characterized by a few common elements : a set of three spacecraft in long - baseline ( mkm ) orbits , monitoring their relative displacements using laser interferometry ; drag - free operation ( except for lagrange ) , whereby displacement measurements are referenced to freely falling test masses protected by the spacecraft , which hover around the masses using precise micro - newton thrusters ; frequency correction of laser noise using a variety of means , including onboard cavities and interferometers , arm locking , and a lisa - specific technique known as time - delay interferometry . the predictions of gr that can be tested by space - based gw observatories include the absence of gravitational fields other than the metric tensor ; the number and character of gw polarization states ; the speed of gw propagation ; the detailed progress of binary inspiral , as driven by nonlinear gravitational dynamics and loss of energy to gws ; the strength and shape of the gws from binary merger and ringdown ; the true nature of astrophysical black holes ; and more . some of these tests will also be performed with ground - based gw detectors and pulsar - timing observations , but space - based tests will almost always have superior accuracy and significance , because low - frequency sources are intrinsically stronger , and will spend a larger time within the band of good detector sensitivity . for binary systems with very asymmetric mass ratios , such as extreme mass - ratio inspirals ( emris ) , lisa - like missions will measure hundreds of thousands of orbital cycles ; because successful detections require matching the phase of signals throughout their evolution , it follows that these observations will be exquisitely sensitive to source parameters . the data - analysis detection problem will be correspondingly delicate , but has been tackled both theoretically , and in a practical program of mock data challenges for lisa [ 37 , 39 , 450 ] . section 2 provides the briefest overview of einstein s gr , of the theoretical framework in which it can be tested , and of a few leading alternative theories . it also introduces the black - hole paradigm , which augments einstein s equations with a few assumptions of physicality that lead to the prediction that the end result of gravitational collapse are black holes described by the kerr metric . section 4 summarizes the main classes of gw sources that would be observed by lisa - like detectors , and that can be used to test gr . section 5 examines the tests of gravitational dynamics that can be performed with these sources , while section 6 discusses the tests of the black - hole nature and structure . ( a conspicuous omission are possible stochastic gw backgrounds of cosmological origin ; indeed , in this article we do not discuss the role of space - based detectors as probes of cosmology and early - universe physics . ) newton s theory of gravitation provided a description of the effect of gravity through the inverse square law without attempting to explain the origin of gravity . the inverse square law provided an accurate description of all measured phenomena in the solar system for more than two hundred years , but the first hints that it was not the correct description of gravitation began to appear in the late 19th century , as a result of the improved precision in measuring phenomena such as the perihelion precession of mercury . einstein s contribution to our understanding of gravity was not only practical but also aesthetic , providing a beautiful explanation of gravity as the curvature of spacetime . in developing gr as a generally covariant theory based on a dynamical spacetime metric , einstein sought to extend the principle of relativity to gravitating systems , and he built on the crucial 1907 insight that the equality of inertial and gravitational mass allowed the identification of inertial systems in homogeneous gravitational fields with uniformly accelerated frames the principle of equivalence . einstein was also guided by his appreciation of ricci and levi - civita s absolute differential calculus ( later to become differential geometry ) , arguably as much as by the requirement to reproduce newtonian gravity in the weak - field limit . indeed , one could say that gr was born of almost pure thought . einstein s theory of gr is described by the action 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{{\rm{gr } } } } = \int { \sqrt { - g } r{{\rm{d}}^4}x},$$\end{document } in which g is the determinant of the spacetime metric and r = gr is the ricci scalar , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${r^{\mu \nu } } = r_{\mu \alpha \nu}^\alpha$\end{document } is the ricci tensor , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$r_{\beta \gamma \delta}^\alpha = \gamma _ { \beta \delta , \gamma}^\alpha - \gamma _ { \beta \gamma , \delta}^\alpha + \gamma _ { \gamma \lambda}^\alpha \gamma _ { \beta \delta}^\lambda - \gamma _ { \delta \lambda}^\alpha \gamma _ { \beta \gamma}^\lambda$\end{document } the riemann curvature tensor , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\gamma _ { \beta \gamma}^\alpha = { g^{\alpha \delta}}({g_{\beta \delta , \gamma } } + { g_{\delta \gamma , \beta } } - { g_{\beta \gamma , \delta}})$\end{document } the affine connection , and a comma denotes a partial derivative . when coupled to a matter distribution , this action yields the field equations 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_{\mu \nu } } \equiv { r_{\mu \nu } } - { 1 \over 2}{g_{\mu \nu}}r = { { 8\pi g } \over { { c^4}}}{t_{\mu \nu}},$$\end{document } where denotes the stress - energy tensor of the matter . since the development of the theory , gr has withstood countless experimental tests [ 471 , 443 , 444 ] based on measurements as different as atomic - clock precision , orbital dynamics ( most notably lunar laser ranging ) , astrometry , and relativistic astrophysics ( most exquisitely the binary pulsar [ 293 , 471 ] , but not only ) . it is therefore the correct and natural benchmark against which to compare alternative theories using future observations and we will follow the same approach in this article . unlike in the case of newtonian gravity at the time that gr was developed , there are no current observations that gr can not explain that can be used to guide development of alternatives.1 nonetheless , there are crucial aspects of einstein s theory that have never been probed directly , such as its strong - field dynamics and the propagation of field perturbations ( gws ) . furthermore , it is known that classical gr must ultimately fail at the planck scale , where quantum effects become important , and traces of the quantum nature of gravity may be accessible at lower energies . as emphasized by will , gr has no adjustable constants , so every test is potentially deadly , and a probe that could reveal new physics . will s living review and his older monograph are the fundamental references about the experimental verification of gr . in this section , we give only a brief overview of what may be called will s standard model for alternative theories of gravity , which proceeds through four steps : a ) strong evidence for the equivalence principle supports a metric formulation for gravity ; b ) metric theories are classified according to what gravitational fields ( scalar , vector , tensor ) they prescribe ; c ) slow - motion , weak - field conservative dynamics are described in a unified parameterized post - newtonian ( ppn ) formalism , and constrained by experiment and observations ; d ) finally , equations for the slow - motion generation and weak - field propagation of gravitational radiation are derived separately for each metric theory , and again compared to observations . many of the tests of gravitational physics envisaged for lisa belong in this last sector of will s standard model , and are discussed in section 5.1 of this review . this scheme however leaves out two other important points of contact between gravitational phenomenology and lisa s gw observations : the strong - field , nonlinear dynamics of black holes and their structure and excitations , especially as probed by small orbiting bodies . we will deal with these in sections 5 and 6 , respectively ; but let us first delve into will s standard model . the equivalence principle and metric theories of gravitation . einstein s original intuition placed the equivalence principle as a cornerstone for the theories that describe gravity as curved spacetime . as formulated by newton , the principle states simply that inertial and gravitational mass are proportional , and therefore all test bodies fall with the same acceleration ( in modern usage , this is known as the weak equivalence principle , or wep ) . dicke later recognized that in developing gr einstein had implicitly posited a broader principle ( einstein s equivalence principle , or eep ) that consists of wep plus local lorentz invariance and local position invariance : that is , of the postulates that the outcome of local non - gravitational experiments is independent of , respectively , the velocity and position of the local freely - falling reference frames in which the experiments are performed . weak equivalence principle ( wep ) : gravitational and inertial masses are equivalent ( neglecting self gravity).2 . einstein equivalence principle ( eep ) : local position invariance and local lorentz invariance apply in addition to the wep.3 . strong equivalence principle ( sep ) : eep applies also for self - gravitating objects . turyshev gives a current review of the experimental verification of wep ( shown to hold to parts in 10 by differential free - fall tests ) , local lorentz invariance ( verified to parts in 10 by clock - anisotropy experiments ) , and local position invariance ( verified to parts in 10 by gravitational - redshift experiments , and to much greater precision when looking for possible time variations of fundamental constants ) . although these three parts of eep appear distinct in their experimental consequences , their underlying physics is necessarily related in any theory of gravity , so schiff conjectured ( and others argued convincingly ) that any complete and self - consistent theory of gravity that embodies wep must also realize eep . eep leads to metric theories of gravity in which spacetime is represented as a pseudo - riemannian manifold , freely - falling test bodies move along the geodesics of its metric , and non - gravitational physics is obtained by applying special - relativistic laws in local freely - falling frames . gr is , of course , a metric theory of gravity ; so are scalar - vector - tensor theories such as brans - dicke theory , which include other gravitational fields in addition to the metric . by contrast , theories with dynamically varying fundamental constants and theories ( such as superstring theory ) that introduce additional wep - violating gravitational fields [ 471 , section 2.3 ] are not metric . neither are most theories that provide short - range and long - range modifications to newton s inverse - square law . the scalar and vector fields in scalar - vector - tensor theories can not directly affect the motion of matter and other non - gravitational fields ( which would violate wep ) , but they can intervene in the generation of gravity and modify its dynamics . these extra fields can be dynamical ( i.e. , determined only in the context of solving for the evolution of everything else ) or absolute ( i.e. , assigned a priori to fixed values ) . the minkowski metric of special relativity is the classic example of absolute field ; such fields may be regarded as philosophically unpleasant by those who dislike feigning hypotheses , but they have a right of citizenship in modern physics as frozen in the additional fields can potentially alter the outcome of local gravitational experiments : while the local gravitational effects of different metrics far away can always be erased by describing physics in a freely - falling reference frame ( which is to say , the local boundary conditions for the metric can be arranged to be flat spacetime ) , the same is not true for scalar and vector fields , which can then affect local gravitational dynamics by their interaction with the metric . this amounts to a violation not of eep , but of the strong equivalence principle ( sep ) , which states that eep is also valid for self - gravitating bodies and gravitational experiments . sep is verified to parts in 10 by combined lunar laser - ranging and laboratory experiments . so far , gr appears to be the only viable metric theory that fully realizes sep . the ppn formalism . because the experimental consequences of different metric theories follow from the specific metric that is generated by matter ( possibly with the help of the extra gravitational fields ) , and because all these theories must realize newtonian dynamics in appropriate limiting conditions , it is possible to parameterize them in terms of the coefficients of a slow - motion , weak - field expansion of the metric . these coefficients appear in front of gravitational potentials similar to the newtonian potential , but involving also matter velocity , internal energy , and pressure . this scheme is the parameterized post - newtonian formalism , pioneered by nordtvedt and extended by will ( see for details ) . of the ten ppn parameters in the current version of the formalism , two are the celebrated and ( already introduced by eddington , robertson , and schiff for the classical tests of gr ) that rule , respectively , the amount of space curvature produced by unit rest mass and the nonlinearity in the superposition of gravitational fields . in gr , and each have the value 1 . the other eight parameters , if not zero , give origin to violations of position invariance ( ) , lorentz invariance ( 13 ) , or even of the conservation of total momentum ( 3 , 14 ) and total angular momentum ( 13 , 14 ) . the ppn formalism is sufficiently accurate to describe the tests of gravitation performed in the solar system , as well as many tests using binary - pulsar observations . the parameter is currently constrained to 1 a few 10 by tests of light delay around massive bodies using the cassini spacecraft ; to 1 a few 10 by lunar laser ranging .2 the other ppn parameters have comparable bounds around zero from solar - system and pulsar measurements , except for 3 , which is known exceedingly well from pulsar observations . tests in the ppn framework have tightly constrained the field of viable alternatives to gr , largely excluding theories with absolute elements that give rise to preferred - frame effects . the ( indirect ) observation of gw emission from the binary pulsar and the accurate prediction of its by einstein s quadrupole formula have definitively excluded other theories [ 471 , 422 ] . yet more gr alternatives were conceived to illuminate points of principle , but they are not well motivated physically and therefore are hardly candidates for experimental verification . some of the theories that are still alive are described in the following . the addition of a single scalar field to gr produces a theory described by the einstein - frame action ( see , e.g. , ) , 3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde i = { ( 16\pi g)^{- 1}}\int { [ \tilde r - 2{{\tilde g}^{\mu \nu}}{\partial _ \mu}\varphi { \partial _ \nu}\varphi - v(\varphi)]{{(- \tilde g)}^{1/2}}{{\rm{d}}^4}x + { i_{{\rm{matter}}}}({\psi _ { \rm{m}}},{a^2}(\varphi){{\tilde g}_{\mu \nu } } ) } , $ $ \end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde g}_{\mu \nu}}$\end{document } is the metric , the ricci curvature scalar \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\tilde r}$\end{document } yields the general - relativistic einstein - hilbert action , and the two adjacent terms are kinetic and potential energies for the scalar field . note that in the action imatter for matter dynamics , the metric couples to matter through the function a( ) , so this representation is not manifestly metric ; it can however be made so by a change of variables that yields the jordan - frame action , 4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$i = { ( 16\pi g)^{- 1}}\int { [ \phi r - { \phi ^{- 1}}\omega ( \phi){g^{\mu \nu}}{\partial _ \mu}\phi { \partial _ \nu}\phi - { \phi ^2}v]{{(- g)}^{1/2}}{{\rm{d}}^4}x + { i_{{\rm{matter}}}}({\psi _ { \rm{m}}},{g_{\mu \nu } } ) } , $ $ \end{document } where a( ) is the transformed scalar field , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${g_{\mu \nu } } \equiv { a^2}(\varphi){{\tilde g}_{\mu \nu}}$\end{document } is the physical metric underlying gravitational observations , and 3 + 2( ) = [ d(ln a())/d ] . brans - dicke theory corresponds to fixing to a constant bd , and it is indistinguishable from gr in the limit bd . in the ppn framework , the only parameter that differs from gr is = ( 1 + bd)/(2 + bd ) . damour and esposito - farese considered an expansion of log a( ) around a cosmological background value , 5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\log a(\varphi ) = { \alpha _ 0}(\varphi - { \varphi _ 0 } ) + { 1 \over 2}{\beta _ 0}{(\varphi - { \varphi _ 0})^2 } + \cdots , $ $ \end{document } where 0 ( and further coefficients ) = 0 reproduces brans - dicke with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\alpha _ 0 ^ 2 = 1/(2{\omega _ { { \rm{bd } } } } + 3),\,{\beta _ 0 } > 0$\end{document } , 0 > 0 causes the evolution of the scalar field toward 0 ( and therefore toward gr ) ; and 0 < 0 may allow a phase change inside objects like neutron stars , leading to large sep violations . these parameters are bound by solar - system , binary - pulsar , and gw observations [ 143 , 186 ] . scalar - tensor theories have found motivation in string theory and cosmological models , and have attracted the most attention in terms of tests with gw observations . the most general second - order action in such a theory takes the form 6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { s = { 1 \over { 16\pi g}}\int { { { \rm{d}}^4}x\sqrt { - g } } \left [ { ( 1 + \omega { u_\mu}{u^\nu})r - k_{\alpha \beta}^{\mu \nu}u_{;\mu}^\alpha u_{;\nu}^\beta + \lambda ( { u_\mu}{u^\mu } + 1 ) } \right ] } , \\ { { \rm{where}}\,\,k_{\alpha \beta}^{\mu \nu } = { c_1}{g^{\mu \nu}}{g_{\alpha \beta } } + { c_2}\delta _ \alpha ^\mu \delta _ _ \beta ^\mu \delta _ \alpha ^\nu - { c_4}{u^\mu}{u^\nu}{g_{\alpha \beta}},\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ \end{array}$$\end{document } in which a semicolon denotes covariant differentiation , and the coefficients ci are arbitrary constants . there are two types of vector - tensor theories : in unconstrained theories , 0 and the constant is arbitrary , while in einstein - aether theories the vector field is constrained to have unit norm , so the lagrange multiplier is arbitrary and the constraint allows to be absorbed into a rescaling of g. for the unconstrained theory , only versions of the theory with c4 = 0 have been studied and for these the field equations are 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { \quad \quad \quad \quad { r_{\kappa \lambda } } - { 1 \over 2}r{g_{\kappa \lambda } } + \omega \theta _ { \kappa \lambda}^{(\omega ) } + \eta \theta _ { \kappa \lambda}^{(\eta ) } + \epsilon \theta _ { \kappa \lambda}^{(\epsilon ) } + \tau \theta _ { \kappa \lambda}^{(\tau ) } + \lambda { g_{\kappa \lambda } } = 0 , } \\ { \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \epsilon f_{\mu \nu}^{;\nu } + { 1 \over 2}\tau u_{\mu ; \nu}^{;\nu } - { 1 \over 2}\omega { u_\mu}r - { 1 \over 2}\eta { u^\alpha}{r_{\mu \alpha } } = 0 , } \\ { \quad \quad \quad \quad \quad \theta _ { \kappa \lambda}^{(\omega ) } = { u_\kappa}{u_\lambda}r + { u^2}{r_{\kappa \lambda } } - { 1 \over 2}{g_{\kappa \lambda}}{u^2}r - { { ( { u^2})}_{;\kappa \lambda } } + { g_{\kappa \lambda}}{\square_g}{u^2 } , } \\ { \theta _ { \kappa \lambda}^{(\eta ) } = 2{u^\alpha}{u_{\left [ \kappa \right.}}{r_{\left . \lambda \right]}}_\alpha - { 1 \over 2}{g_{\kappa \lambda}}{u^\alpha}{u^\beta}{r_{\alpha \beta } } - { { ( { u^\alpha}{u_{\left [ \kappa \right.}})}{}_{\left . { ; \lambda } \right]\alpha } } + { 1 \over 2}{\square_g}({u_\kappa}{u_\lambda } ) + { 1 \over 2}{g_{\kappa \lambda}}{{({u^\alpha}{u^\beta})}_{;\alpha \beta}},\quad \quad \quad \quad \,\ , } \\ { \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \,\,\,\,\theta _ { \kappa \lambda}^{(\epsilon ) } = - 2(f_\kappa ^\alpha { f_{\lambda \alpha } } - { 1 \over 4}{g_{\kappa \lambda}}{f_{\alpha \beta}}{f^{\alpha \beta } } ) , } \\ { \theta _ { \kappa \lambda}^{(\tau ) } = { u_{\kappa ; \alpha}}u_\lambda ^{;\alpha } + { u_{\alpha ; \kappa}}u_{;\lambda}^\alpha - { 1 \over 2}{g_{\kappa \lambda}}{u_{\alpha ; \beta}}{u^{\alpha ; \beta } } + { { ( { u^\alpha}{u_{\left [ { \kappa ; \lambda } \right ] } } - u_{;\left [ \kappa \right.}^\alpha { u_{\left . \lambda \right ] } } - { u_{\left [ \kappa \right.}}u_{\left . \lambda \right]}^{;\alpha})}_{;\alpha } } , } \\ \end{array}$$\end{document } where f = u; u; , u uu , = c2 , = (c2 + c3)/2 , and = (c1 + c2 + c3 ) . we use the usual subscript notation , such that ( , ) and [ , ] denote symmetric and antisymmetric sums . in the constrained einstein - aether theory the field equations are 8\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { j^\alpha}_{\mu ; \alpha } - { c_4}{{\dot u}^\alpha}u_{;\mu}^\alpha = \lambda { u_\mu},\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \,\,\ , } \\ { { \rm{where}}\,{j^\alpha}_\mu = k_{\mu \nu}^{\alpha \beta}u_{;\beta}^\nu , \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { { g_{\alpha \beta } } = t_{\alpha \beta}^{(u ) } + { { 8\pi g } \over { { c^4}}}t_{\alpha \beta}^{{\rm{matter}}},\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { t_{\alpha \beta}^{(u ) } \equiv { { \left({{j_{\left(\alpha \right.}}^\mu { u_{\left . \beta \right ) } } - { j_{\left({\alpha \beta } \right)}}{u^\mu } } \right)}_{;\mu } } + { c_1}\left({u_{;\mu}^\alpha u_\beta ^{;\mu } - { u_{\mu ; \alpha}}u_{;\beta}^\mu } { \quad \quad \quad + \left [ { { u_\nu}j_{;\mu}^{\mu \nu } - { c_4}{{\dot u}^2 } } \right]{u_\alpha}{u_\beta } - { 1 \over 2}{g_{\alpha \beta}}{l_u } , } \\ \end{array}$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\dot u^\alpha } \equiv { u^\beta}u_{;\beta}^\alpha , \,{l_u } = - k_{\mu \nu}^{\alpha \beta}\,u_{;\alpha}^\nu u_{;\beta}^\nu$\end{document } is the aether lagrangian , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$t_{\alpha \beta}^{{\rm{matter}}}$\end{document } is the usual matter stress - energy tensor . via field redefinition this theory can be shown to be equivalent to gr if c1 + c4 = 0 , c1 + c2 + c3 = 0 , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${c_3 } = \pm \sqrt { { c_1}({c_1 } - 2)}$\end{document } . field redefinition can also be used to set c1 + c3 = 0 ; if this constraint is imposed then equivalence to gr is only achieved if the ci are all zero . this constraint is therefore appropriate to pose einstein - aether theory as an alternative to test against gr , since then any non - zero values of the ci would represent genuine deviations from gr . unconstrained vector - tensor theories were introduced in the 1970s as a straw - man alternative to gr , but they have four arbitrary parameters and leave the magnitude of the vector field unconstrained , which is a serious defect . interest in einstein - aether theories was prompted by the desire to construct a covariant theory that violated lorentz invariance under boosts , by having a preferred reference frame the aether , represented by the vector u. the preferred reference frame also provides a universal notion of time . interest in theories that violate lorentz symmetry has recently been revived as a possible window onto aspects of quantum gravity . the natural extension of scalar - tensor and vector - tensor theories are scalar - vector - tensor theories in which the gravitational field is coupled to a vector field and one or more scalar fields . these theories are relativistic generalizations of modified newtonian dynamics ( mond ) , which was proposed in order to reproduce observed rotation curves on galactic scales . the action takes the form 9\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s = { 1 \over { 16\pi}}\int { { { \rm{d}}^4}x } \sqrt { - g } ( { l_g } + { l_s } + { l_u } + { l_{{\rm{matter}}}}),$$\end{document } where lg = ( r 2)/g and lmatter are the usual gravitational and matter lagrangians . there are two main versions of the theory , which differ in the choice of the scalar - field and vector - field lagrangians ls and lu . in tensor - vector - scalar gravity ( teves ) the dynamical vector field u is coupled to a dynamical scalar field . a second scalar field is here considered non - dynamical . the lagrangians are 10\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { l_s } = { 1 \over { 2g}}\left [ { { \sigma ^2}\left({{g^{\alpha \beta } } - { u^\alpha}{u^\beta } } \right){\phi _ { , \alpha}}{\phi _ { , \beta } } + { 1 \over 2}{g \over { { l^2}}}{\sigma ^4}f(g{\sigma ^2 } ) } \right ] , } \\ { { l_u } = { k \over { 2g}}\left [ { { g^{\alpha \beta}}{g^{\mu \nu}}{b_{\alpha \mu}}{b_{\beta \nu } } + 2{\lambda \over k}({u^\mu}{u_\mu } + 1 ) } \right],\quad \quad \quad \,\ , } \\ \end{array}$$\end{document } where b = u, u, , f is an unspecified dimensionless function , k is a dimensionless parameter , and l is a constant length parameter . the lagrange multiplier is spacetime dependent , set to enforce normalization of the vector field uu = 1 . in teves the physical metric that governs the gravitational dynamics of ordinary matter does not coincide with g , but is determined by the scalar field through 11\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\hat g_{\mu \nu } } = { e^{2\phi}}{g_{\mu \nu } } - 2{u_\mu}{u_\nu}\sinh ( 2\phi){.}$$\end{document } an alternative version of teves , called bi - scalar - tensor - vector gravity ( bstv ) has also been proposed , in which the scalar field is allowed to be dynamical . teves is able to explain galaxy rotation curves and satisfies constraints from cosmology and gravitational lensing , but stars are very unstable and the bullet cluster observations ( which point to dark matter ) can not be explained . in scalar - tensor - vector gravity ( stvg ) the lagrangian for the vector field is taken to be 12\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${l_u } = \omega \left [ { { b^{\mu \nu}}{b_{\mu \nu } } - 2{\mu ^2}{u^\mu}{u_\mu } + { v_u}(u ) } \right],$$\end{document } with b defined as before . the three constants , , and g that enter this action and the gravitational action are then taken to be scalar fields governed by the lagrangian 13\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${l_s } = { { 16\pi } \over g}\left [ { { 1 \over 2}{g^{\nu \rho}}\left({{{{g_{,\nu}}{g_{,\rho } } } \over { { g^2 } } } + { { { \mu _ { , \nu}}{\mu _ { , \rho } } } \over { { \mu ^2 } } } - { \omega _ { , \nu}}{\omega _ { , \rho } } } \right ) + { { { v_g}(g ) } \over { { g^2 } } } + { { { v_\mu}(\mu ) } \over { { \mu ^2 } } } + { v_\omega}(\omega ) } \right]{.}$$\end{document } it is claimed that stvg predicts no deviations from gr on the scale of the solar system or for small globular clusters , and that it can reproduce galactic rotation curves , gravitational lensing in the bullet cluster , and a range of cosmological observations . it has been proposed that an extension of the esa - led lisa pathfinder technology - demonstration mission may allow additional constraints on this class of theories . to date this theory is derived by replacing r with an arbitrary function f(r ) in the einstein - hilbert action . the action is extremized with respect to the metric coefficients only , and the connection is taken to be the metric connection , depending on the metric components in the standard way . the resulting field equations are 14\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$(- { r_{;\kappa}}{r_{;\lambda } } + { g_{\kappa \lambda}}{r_{;\mu}}{r^{;\mu}})f^{\prime\prime\prime}(r ) + ( - { r_{;\kappa \lambda } } + { g_{\kappa \lambda}}{\square}r)f^{\prime\prime}(r ) + { r_{\kappa \lambda}}f^{\prime}(r ) - { 1 \over 2}{g_{\kappa \lambda}}f(r ) = 0{.}$$\end{document } in the palatini formalism , the field equations are found by extremizing the action over both the metric and the connection . for an f(r ) action the resulting equations are 15\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { r_{\kappa \lambda}}{f^\prime}(r ) - { 1 \over 2}{g_{\kappa \lambda}}f(r ) = 0 , } & { { \nabla _ \alpha}\left [ { \sqrt { - g } { f^\prime}(r){g^{\kappa \lambda } } } \right ] = 0{. } } \\ \end{array}$$\end{document } if the second derivative f(r ) 0 , metric f(r ) gravity can be shown to be equivalent to a brans - dicke theory with bd = 0 , while palatini f(r ) gravity is equivalent to a brans - dicke theory with bd = 3/2 , with no constraint imposed on f(r ) [ 180 , 419 , 146 ] . in both cases , f(r ) theories have attracted a lot of interest in a cosmological context , since the flexibility in choosing the function f(r ) allows a wide range of cosmological phenomena to be described [ 336 , 108 ] , including inflation [ 423 , 459 ] and late - time acceleration [ 107 , 112 ] , without violating constraints from big - bang nucleosynthesis . however , metric f(r ) theories are strongly constrained by solar - system and laboratory measurements if the scalar degree of freedom is assumed to be long - ranged , which modifies the form of the gravitational potential . this problem can be avoided by assuming a short - range scalar field , but then f(r ) theories can only explain the early expansion of the universe and not late - time acceleration . the chameleon mechanism has been invoked to circumvent this , as it allows the scalar - field mass to be a function of curvature , so that the field can be short ranged within the solar system but long ranged on cosmological scales . for example , in palatini f(r ) gravity the post - newtonian metric depends on the local matter density , while in metric f(r ) gravity with f(r ) < 0 there is a ricci - scalar instability that arises because the effective gravitational constant increases with increasing curvature , leading to a runaway instability for small stars [ 56 , 55 ] . we refer the reader to [ 419 , 146 ] for more complete reviews of the current understanding of f(r ) gravity . yunes and others [ 6 , 8 , 12 , 13 , 103 , 104 , 111 , 158 , 189 , 212 , 218 , 272 , 323 , 416 , 488 , 491 , 496 , 501 , 503 , 499 , 340 ] have recently developed an extensive analysis of the observational consequences of jackiw and pi s chern - simons gravity , which extends the hilbert action with an additional pontryagin term * rr that is quadratic in the riemann tensor : 16\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$i = { ( 16\pi g)^{- 1}}\int { [ \tilde r - { 1 \over 4}\theta ^{\ast}rr]{{(- g)}^{1/2}}{{\rm{d}}^4}x + { { { \rm i } } _ { { \rm{matter}}}}({\psi _ { \rm{m}}},{g_{\mu \nu}});}$$\end{document } here \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${}^*rr = { } ^*{r^a}_b{}^{cd}{r^b}_{acd}$\end{document } is built with the help of the dual riemann tensor \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$^{\ast}{r^a}_b{}^{cd } = { 1 \over 2}{\epsilon ^{cde\,f}}{r^a}_{be\,f}$\end{document } , and it can be expressed as the divergence of the gravitational chern - simons topological current;3 the scalar field can be treated either as a dynamical quantity , or an absolute field . in both cases , * rr vanishes , either dynamically , or as a constraint on acceptable solutions , needed to enforce coordinate - invariant matter dynamics , which restricts the space of solutions available to gr . chern - simons gravity is motivated by string theory and by the attempt to develop a quantum theory of gravity satisfying a gauge principle . the pontryagin term arises in the standard model of particle physics as a gauge anomaly : the classical gravitational noether current that comes from the symmetry of the gravitational action is no longer conserved when the theory is quantized , but has a divergence proportional to the pontryagin term . this anomaly can be canceled by modifying the action via the addition of the chern - simons pontryagin term . the same type of correction arises naturally in string theory through the green - schwarz anomaly - canceling mechanism , and in loop quantum gravity to enforce parity and charge - parity conservation . the presence of the chern - simons correction leads to parity violation , which has various observable consequences , with magnitude depending on the chern - simons coupling , which string theory predicts will be at the planck scale . if so , these effects will never be observable , but various mechanisms have been proposed that could enhance the strength of the chern - simons coupling , such as non - perturbative instanton corrections , fermion interactions , large intrinsic curvatures or small string couplings at late times . for further details on all aspects of chern - simons gravity , this theory arises by adding to the action all possible terms that are quadratic in the ricci scalar , ricci tensor , and riemann tensor . for the action 17\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s = { 1 \over { 16\pi g}}\int { \sqrt { - g } ( - 2\lambda + r + \alpha { r^2 } + \beta { r_{\sigma \tau}}{r^{\sigma \tau } } + \gamma { r_{\alpha \beta \gamma \delta}}{r^{\alpha \beta \gamma \delta}}){{\rm{d}}^4}x}$$\end{document } the field equations are 18\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { r_{\kappa \lambda } } - { 1 \over 2}r{g_{\kappa \lambda } } + \alpha { k_{\kappa \lambda } } + \beta { l_{\kappa \lambda } } + \lambda { g_{\kappa \lambda } } = 0,\,\,\,\,\,\,\,\,\,\,\quad \quad \quad \quad \,\,\ , } \\ { { k_{\kappa \lambda } } = - 2{r_{;\kappa \lambda } } + 2{g_{\kappa \lambda}}{\square}r - { 1 \over 2}{r^2}{g_{\kappa \lambda } } + 2r{r_{\kappa \lambda}},\quad \quad \quad \quad \,\,\,\ , } \\ { { l_{\kappa \lambda } } \equiv - 2r_{\kappa ; \sigma \lambda}^\sigma + { \square}{r_{\kappa \lambda } } + { 1 \over 2}{g_{\kappa \lambda}}{\square}r - { 1 \over 2}{g_{\kappa \lambda}}{r^{\sigma \tau } } + 2r_\kappa ^\sigma { r_{\sigma \lambda}}{. } } \\ \end{array}$$\end{document } this class of theories is parameterized by the coefficients , , and . more recently , stein and yunes considered a more general form of quadratic gravity that includes the pontryagin term from chern - simons gravity . their action was 19\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { s \equiv \int { \sqrt { - g } } \left\{{\kappa r + { \alpha _ 1}{f_1}(\theta){r^2 } + { \alpha _ 2}{f_2}(\theta){r_{ab}}{r^{ab } } + { \alpha _ 3}{f_3}(\theta){r_{abcd}}{r^{abcd } } } \right . } \\ { + { \alpha _ 4}{f_4}(\theta)r_{abcd}^{\ast } { r^{abcd } } - \left . { { \beta \over 2}[{\nabla _ a}\theta { \nabla ^a}\theta + 2v(\theta ) ] + { { \mathcal l}_{{\rm{matter } } } } } \right\},\quad } \\ \end{array}$$\end{document } in which the i and are coupling constants , is a scalar field , and matter is the matter lagrangian density as before . there are two versions of this theory : a non - dynamical version in which the functions are constants , and a dynamical version in which they are not . general quadratic theories are known to exhibit ghost fields negative mass - norm states that violate unitarity ( see , e.g. , for a discussion and further references ) . these occur generically , although models with an action that is a function only of r and r 4r r + r r only are ghost - free . ghost fields are also present in chern - simons modified gravity [ 323 , 158 ] , which places strong constraints on the parameters of that model . massive - graviton theories were first considered by pauli and fierz [ 350 , 175 , 176 ] , whose theory is generated by an action of the form 20\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { s_{{\rm{pf } } } } = m_p^2\int { { { \rm{d}}^4}x } \left [ { - { 1 \over 4}{{({\partial _ \mu}{h_{\nu \rho}})}^2 } + { 1 \over 4}{{({\partial _ \mu}h)}^2 } - { 1 \over 2}({\partial _ \mu})({\partial ^\nu}h_\nu ^\mu ) + { 1 \over 2}({\partial _ \mu}{h_{\nu \rho}})({\partial ^\nu}{h^{\mu \rho } } ) } \right . } \\ { \left . { - { 1 \over 4}{m^2}({h_{\mu \nu}}{h^{\mu \nu } } - { h^2 } ) + m_p^{- 2}{t_{\mu \nu}}{h^{\mu \nu } } } \right ] , } \\ \end{array}$$\end{document } in which h is a rank - two covariant tensor , m and mp are mass parameters , t is the matter energy - momentum tensor , indices are raised and lowered with the minkowski metric , and h = h. the terms on the first line of this expression are generated by expanding the einstein - hilbert action to quadratic order in h. the massive graviton term is m ( hh h ) ; it contains a spin-2 piece h and a spin-0 piece h. this model suffers from the van dam - velten - zakharov discontinuity [ 454 , 505 ] : no matter how small the graviton mass , the pauli - fierz theory leads to different physical predictions from those of linearized gr , such as light bending . the theory also predicts that the energy lost into gws from a binary is twice the gr prediction , which is ruled out by current binary - pulsar observations . it might be possible to circumvent these problems and recover gr in the weak - field limit by invoking the vainshtein mechanism [ 446 , 41 ] , which relies on nonlinear effects to hide certain degrees of freedom for source distances smaller than the vainshtein radius . the massive graviton can therefore become effectively massless , recovering gr on the scale of the solar system and in binary - pulsar tests , while retaining a mass on larger scales . in such a scenario , the observational consequences for gws would be a modification to the propagation time for cosmological sources , but no difference in the emission process itself . there are also non - pauli - fierz massive graviton theories . for these , the action is the same as that in eq . ( 20 ) , but the first term on the second line ( the massive graviton term ) takes the more general form 21\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$- ( { k_1}{h_{\mu \nu}}{h^{\mu \nu } } + { k_2}{h^2}),$$\end{document } where k1 and k2 are new constants of the theory that represent the squared masses of the spin-2 and spin-0 gravitons respectively . this theory can recover gr in the weak field , since k1 and k2 can independently be taken to zero , with modifications to weak - field effects that are on the order of the graviton mass squared . these theories are generally thought to suffer from instabilities [ 350 , 175 , 176 ] , which arise because the spin-0 graviton carries negative energy . however , it was shown in that the spin-0 graviton can not be emitted without spin-2 gravitons also being generated . the spin-2 graviton energy is positive and greater than that of spin-2 gravitons in gr , which compensates for the spin-0 graviton s negative energy . the total energy emitted is therefore always positive , and it converges to the gr value in the limit that the spin-2 graviton mass goes to zero . these alternative massive - graviton theories are therefore perfectly compatible with current observational constraints , but make very different predictions for strong gravitational fields , including the absence of horizons for black - hole spacetimes and oscillatory cosmological solutions . despite these potential problems , the existence of a massive graviton can be used as a convenient strawman for gw constraints , since the speed of gw propagation can be readily inferred from gw observations and compared to the speed of light . these proposed tests generally make no reference to an underlying theory but require only that the graviton has an effective mass and hence gws suffer dispersion . as their name suggests , there are two metrics in bimetric theories of gravity [ 382 , 384 ] . one is dynamical and represents the tensor gravitational field ; the other is a metric of constant curvature , usually the minkowski metric , which is non - dynamical and represents a prior geometry . rosen s theory has the action [ 381 , 382 , 383 , 384 ] 22\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s = { 1 \over { 64\pi g}}\int { { { \rm{d}}^4}x } \left [ { \sqrt { - \det ( \eta ) } { \eta ^{\mu \nu}}{g^{\alpha \beta}}{g^{\gamma \delta}}\left({{g_{\alpha \gamma { \vert}\mu}}{g_{\alpha \delta { \vert}\nu } } - { 1 \over 2}{g_{\alpha \gamma { \vert}\mu}}{g_{\alpha \delta { \vert}\nu } } } \right ) } \right ] + { s_{{\rm{matter}}}},$$\end{document } in which is the fixed flat , non - dynamical metric , g is the dynamical gravitational metric and the vertical line in subscripts denotes a covariant derivative with respect to . the final term , smatter , denotes the action for matter fields . the field equations may be written 23\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\square}_\eta}{g_{\mu \nu } } - { g^{\alpha \beta}}{\eta ^{\gamma \delta}}{g_{\mu \alpha { \vert}\gamma}}{g_{\nu \beta { \vert}\delta } } = - 16\pi g\sqrt { \det ( g)/\det ( \eta ) } \left({{t_{\mu \nu } } - { 1 \over 2}{g_{\mu \nu}}t } \right){.}$$\end{document } lightman and lee developed a bimetric theory based on a non - metric theory of gravity due to belinfante and swihart . the action for this bsll theory is 24\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s = { 1 \over { 64\pi g}}\int { { { \rm{d}}^4}x\sqrt { - \det ( \eta ) } \left({{1 \over 4}{h_{\mu \nu { \vert}\alpha}}{h^{\mu \nu { \vert}\alpha } } - { 5 \over { 64}}{h_{,\alpha}}{h^{,\alpha } } } \right ) + { s_{{\rm{matter}}}},}$$\end{document } in which is the non - dynamical flat background metric and is a dynamical gravitational tensor related to the gravitational metric g via 25\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { g_{\mu \nu } } = { { \left({1 - { 1 \over { 16}}h } \right)}^2}\delta _ \mu ^\alpha { \delta _ { \alpha \nu } } , } \\ { \delta _ \nu ^\mu = \delta _ \nu ^\alpha \left({\delta _ \alpha ^\mu - { 1 \over 2}h_\mu ^\alpha } \right),\,\,\ , } \\ \end{array}$$\end{document } in which \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta _ \nu ^\mu$\end{document } is the kronecker delta and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\delta _ indices on and h are raised and lowered with , but on all other tensors indices are raised and lowered by g. both the rosen and bsll bimetric theories give rise to alternative gw polarization states , and have been used to motivate the construction of the parameterized post - einsteinian ( ppe ) waveform families discussed in section 5.2.2 . there is also a bimetric theory due to rastall , in which the metric is an algebraic function of the minkowski metric and of a vector field k. the action is 26\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$s = { 1 \over { 64\pi g}}\int { { { \rm{d}}^4}x } \left [ { \sqrt { - \det ( g ) } f(n){k_{\mu ; \nu}}{k^{\mu ; \nu } } } \right ] + { s_{{\rm{matter}}}},$$\end{document } in which f(n ) = n/(n + 2 ) , n = gkk and a semicolon denotes a derivative with respect to the gravitational metric g. the metric follows from k by way of 27\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${g_{\mu \nu } } = \sqrt { 1 + { \eta ^{\alpha \beta}}{k_\alpha}{k_\beta } } ( { \eta _ { \mu \nu } } + { k_\mu}{k_\nu}),$$\end{document } where is again the non - dynamical flat metric . this theory has not been considered in a gw context and we will not mention it further ; more details , including the field equations , can be found in . the present consensus is that all of the compact objects observed to reside in galactic centers are supermassive black holes , described by the kerr metric of gr . this explanation follows naturally in gr from the black - hole uniqueness theorems and from a set of additional assumptions of physicality , briefly discussed below . if a deviation from kerr is inferred from gw observations , it would imply that the assumptions are violated , or possibly that gr is not the correct theory of gravity . space - based gw detectors can test black - hole kerr - ness by measuring the gws emitted by smaller compact bodies that move through the gravitational potentials of the central objects ( see section 6.2 ) . kerr - ness is also tested by characterizing multiple ringdown modes in the final black hole resulting from the coalescence of two precursors ( see section 6.3 ) . the current belief that kerr black holes are ubiquitous follows from work on mathematical aspects of gr in the middle of the 20th century . oppenheimer and snyder demonstrated that a spherically - symmetric , pressure - free distribution would collapse indefinitely to form a black hole . this result was assumed to be a curiosity due to spherical symmetry , until it was demonstrated by penrose and by hawking and penrose that singularities arise inevitably after the formation of a trapped surface during gravitational collapse . around the same time , it was proven that the black - hole solutions of schwarschild and kerr are the only static and axisymmetric black - hole solutions in gr [ 248 , 114 , 379 ] . the assumptions that underlie the proof of the uniqueness theorem are that the spacetime is a stationary vacuum solution , that it is asymptotically flat , and that it contains an event horizon but no closed timelike curves ( ctcs ) exterior to the horizon . the lack of ctcs is needed to ensure causality , while the requirement of a horizon is a consequence of the cosmic - censorship hypothesis ( cch ) . the cch embodies this belief by stating that any singularity that forms in nature must be hidden behind a horizon ( i.e. , can not be naked ) , and therefore can not affect the rest of the universe , which would be undesirable because gr can make no prediction of what happens in its vicinity . however , the cch and the non - existence of ctcs are not required by einstein s equations , and so they could in principle be violated . besides the kerr metric , we know of many other black - hole - like solutions to einstein s equations : these are vacuum solutions with a very compact central object enclosed by a high - redshift surface . in fact , any metric can become a solution to einstein s equation : it is sufficient to insert it in the einstein tensor , and postulate the resulting matter however , such matter distributions will not in general satisfy the energy conditions ( see , e.g. , ): the weak energy condition is the statement that all timelike observers in a spacetime measure a non - negative energy density , tvv 0 , for all future - directed timelike vectors v. the null energy condition modifies this condition to null observers by replacing by an arbitrary future - directed null vector k.the strong energy condition requires the ricci curvature measured by any timelike observer to be non - negative , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$({t_{\mu \nu } } - t_\alpha ^\alpha { g_{\mu \nu}}/2){\upsilon ^\mu}{\upsilon ^\nu } \ge 0$\end{document } , for all timelike v.the dominant energy condition is the requirement that matter flow along timelike or null world lines : that is , that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$- t_\nu ^\mu { \upsilon ^\nu}$\end{document } be a future - directed timelike or null vector field for any future - directed timelike vector v. these conditions make sense on broad physical grounds ; but even after imposing them , there remain several black - hole - like solutions besides kerr . thus , space - based gw detectors offer an important test of the black - hole paradigm that follows from gr plus cch , ctc non - existence , and the energy conditions . this paradigm is especially important : putative black holes are observed to be ubiquitous in the universe , so their true nature has significant implications for our understanding of astrophysics . the weak energy condition is the statement that all timelike observers in a spacetime measure a non - negative energy density , tvv 0 , for all future - directed timelike vectors v. the null energy condition modifies this condition to null observers by replacing by an arbitrary future - directed null vector k. the strong energy condition requires the ricci curvature measured by any timelike observer to be non - negative , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$({t_{\mu \nu } } - t_\alpha ^\alpha { g_{\mu \nu}}/2){\upsilon ^\mu}{\upsilon ^\nu } \ge 0$\end{document } , for all timelike v. the dominant energy condition is the requirement that matter flow along timelike or null world lines : that is , that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$- t_\nu ^\mu { \upsilon ^\nu}$\end{document } be a future - directed timelike or null vector field for any future - directed timelike vector v. if one or many non - kerr metrics are found , the hope is that observations will allow us to tease apart the various possible explanations : does the spacetime contain matter , such as an accretion disk , exterior to the black hole?are the cch , the no - ctc assumption , or the energy conditions violated?is the central object an exotic object , such as a boson star [ 389 , 261]?is gravity coupled to other fields ? this can lead to different black - hole solutions [ 265 , 396 , 413 ] , although some such solutions are known ) or suspected to be unstable to generic perturbations.is the theory of gravity just different from gr ? for instance , in chern - simons gravity black holes ( to linear order in spin ) differ from kerr in their octupole moment , and this correction may produce the most significant observational signature in gw observations . while these questions are challenging , we can learn a lot by testing black - hole structure with space - based gw detectors . does the spacetime contain matter , such as an accretion disk , exterior to the black hole ? are the cch , the no - ctc assumption , or the energy conditions violated ? is the central object an exotic object , such as a boson star [ 389 , 261 ] ? this can lead to different black - hole solutions [ 265 , 396 , 413 ] , although some such solutions are known ) or suspected to be unstable to generic perturbations . for instance , in chern - simons gravity black holes ( to linear order in spin ) differ from kerr in their octupole moment , and this correction may produce the most significant observational signature in gw observations . the experimental search for gws began in the 1960s with joseph weber s resonant bars ( and resonant claims ) ; it has since grown into an extensive international endeavor that has produced a network of km - scale gw interferometers ( ligo , ego / virgo , geo600 , and tama / kagra ) , the proposals for space - based observatories such as lisa , and the effort to detect gws by using an array of pulsars as reference clocks . in this section we briefly describe the architecture of lisa - like space - based gw observatories , beginning with the classic lisa design , and then discussing the variations studied in the 20112012 esa and nasa studies [ 20 , 215 ] . we also discuss proposals for detectors that would operate in a higher frequency band ( between 0.1 and 10 hz ) that would bridge the gap in sensitivity between lisa - like and ground - based observatories . the most cited lisa reference is perhaps the 1998 pre - phase a mission study ; a more up - to - date review of the lisa technical architecture is given by jennrich , while the lisa science - case document describes the state of lisa science at the end of the 2000s . here we give only a quick review of the elements of the mission , referring the reader to those references for in - depth discussions . lisa consists of three identical cylindrical spacecraft , approximately 3 m wide and 1 m high , that are launched together and , after a 14-month cruise , settle into an earth - like heliocentric orbit , 20 behind the earth . the orbit of each spacecraft is tuned slightly differently , resulting in an equilateral - triangle configuration with 5 10 km arms ( commensurate with the frequencies of the lisa sensitivity band ) , inclined by 60 with respect to the ecliptic . this configuration is maintained to 1% by orbital dynamics alone for the lifetime of the mission , nominally 5 years , with a goal of 10 . in the course of a year , the center of the lisa triangle completes a full revolution on the ecliptic , while the triangle itself , as well as its normal vector , rotate through 360. lisa detects gws by monitoring the fluctuations in the distances between freely - falling reference bodies in lisa , platinum - gold test masses housed and protected by the spacecraft . lisa uses three pairs of laser interferometric links to measure inter - spacecraft distances with errors near 10 hz . the phase shifts accumulated along different interferometer paths are proportional to an integral of gw strain along those trajectories . the test masses suffer from residual acceleration noise at a level of 3 10 m s , which is dominant below 3 mhz . together , the position and acceleration noises ( divided by a multiple of the armlength ) determine the lisa sensitivity to gw strain , which reaches 10 hz at frequencies of a few mhz . alternatively ( but equivalently ) , we may describe lisa as measuring the gw - induced relative doppler shifts between the local lasers on each spacecraft and the remote lasers . these doppler shifts are directly proportional to a difference of instantaneous gw strains ( as experienced by the local spacecraft at the time of measurement , and by the distant spacecraft at a time retarded by the lisa armlength divided by the speed of light ) . in either description , the 1% breathing of the lisa constellation occurs at frequencies that are safely below the lisa measurement band . the distance measurement between the test masses along each arm is in fact split in three : an inter - spacecraft measurement between the two optical benches , and two local measurements between each test mass and its optical bench . to achieve the inter - spacecraft measurement , 2 w of 1064-nm light the diffracted beams deliver only 100 pw of light to the distant spacecrafts , so they can not be reflected directly ; instead , the laser phase is measured and transponded back by modulating the frequency of the local lasers . each of the two optical assemblies on each spacecraft include the telescope and a zerodur optical bench with bonded fused - silica optical components , which implements the interspacecraft and local interferometers , as well as a few auxiliary interferometric measurements , which are needed to monitor the stability of the telescope structure , to control point - ahead corrections , and to compare the two lasers on each spacecraft . the lisa phasemeters digitize the signals from the optical - bench photodetectors at 50 mhz , and multiply them with the output of local oscillators , computing phase differences and driving the oscillators to track the frequency of the measured signal . this heterodyne scheme is needed to handle doppler shifts ( as well as laser frequency offsets ) as large as 15 mhz . because intrinsic fluctuations in the laser frequencies are indistinguishable from gws in the lisa output , laser frequency noise needs to be suppressed by several orders of magnitude , using a hierarchy of techniques : the lasers are prestabilized to local frequency references ; arm locking may be used to further stabilize the lasers using the lisa arms ( or their differences ) as stable references ; finally , time - delay interferometry is applied in post processing to remove residual laser frequency noise by algebraically combining appropriately - delayed single - link measurements , in such a way that all laser - frequency - noise terms appear as canceling pairs in the combination . with this final step , the lisa measurements combine into synthesized - interferometer observables analogous to the readings of ground - based interferometers such as ligo . the lisa disturbance reduction system ( drs ) minimizes the deviations of the test masses from free - fall trajectories , by shielding them from solar radiation pressure and interplanetary magnetic fields . on each spacecraft , the drs includes two gravitational reference sensors ( grss ) : a grs consists of a 2-kg test mass , enclosed in an electrode housing with capacitive reading and control of test - mass position and orientation , and accompanied by additional components to cage and reposition the test mass , to maintain vacuum , and to control the accumulation of charge . the other crucial part of the drs are the micro - newton thrusters ( on each spacecraft , three clusters of four colloid or field - emission - electric propulsion systems ) , which are controlled in response to the grs readings to maintain the nominal position of the test mass with respect to the spacecraft . in addition to this active correction , test - mass acceleration noise is minimized by the accurate knowledge and correction of spacecraft self - gravity , by enforcing magnetic cleanliness , and by controlling thermal fluctuations . a version of the lisa drs with slightly lower performance will be flown and tested in lisa pathfinder [ 305 , 25 ] , a single - spacecraft technology precursor mission . the lisa response to gws . compared to ground - based interferometers , the lisa response to gws is both richer and more complex [ 166 , 462 , 448 , 133 ] . first , the revolution and rotation of the lisa constellation imprint a sky - position - dependent signature on long - lasting gw signals ( which for lisa include all binary signals ) : the revolution causes a time - dependent doppler shift with a period of a year , and a fractional amplitude of 10 ; the rotation introduces a 1-year periodicity in the lisa equivalent of the ground - based - interferometer antenna patterns , endowing an incoming monochromatic gw signal with eight sidebands , separated by yr = 3.17 10 hz . thus , gw signals as measured by lisa carry information regarding the position of the source ; on the other hand , data analysis must account for these effects , possibly including sky position among the parameters of matched - filtering search templates . second , the long - wavelength approximation , whereby the entire interferometer shrinks and expands as one , can not be used throughout the lisa band ; indeed , the wavelength of gws reaches the lisa armlength at a frequency of 60 mhz . as a consequence , the lisa response to a few - second impulsive gw is not a single pulse , but a collection of pulses with amplitudes and separations dependent on the sky position and polarization of the source ; the effect on high - frequency chirping signals is more subtle , but still present . this further complicates data analysis , and introduces an independent mechanism ( a triangulation of sorts ) to localize gw sources of short duration and high frequency . third , lisa is in effect three detectors in one : this can be understood most easily by considering that subsets of the three lisa arms form three separate michelson - like interferometers ( known in lisa lingo as x , y , and z ) at 120 angles . more formally , the lisa interferometric measurements can be combined into many different tdi observables ( see figure 1 ) , some resembling actual optical setups , others quite exotic , although at most three observables are independent in the sense that any other observable can be reconstructed by time - delaying and summing a generic basis of three observables . furthermore , such a basis can be chosen so that its components have uncorrelated noises , much like widely separated ground - based detectors . one of these must correspond , in effect , to x+y + z , and by symmetry it must be relatively insensitive to gws in the long - wavelength limit , providing for an independent measurement of a combination of instrument noises . lisa - like detectors measure gws by transmitting laser light between three spacecraft in triangular configuration , and comparing the optical phase of the incident lasers against reference lasers on each spacecraft . to avoid extreme requirements on laser - frequency stability over the course of the many seconds required for transmission around the triangle , data analysts will generate time - delayed linear combinations of the phase comparisons ; the combinations simulate nearly equal - delay optical paths around the sides of the triangle , and ( much like an equal - arm michelson interferometer ) they suppress laser frequency noise . many such combinations , including those depicted here , are possible , but altogether they comprise at most three independent gravitational - wave observables . lisa - like detectors measure gws by transmitting laser light between three spacecraft in triangular configuration , and comparing the optical phase of the incident lasers against reference lasers on each spacecraft . to avoid extreme requirements on laser - frequency stability over the course of the many seconds required for transmission around the triangle , data analysts will generate time - delayed linear combinations of the phase comparisons ; the combinations simulate nearly equal - delay optical paths around the sides of the triangle , and ( much like an equal - arm michelson interferometer ) they suppress laser frequency noise . many such combinations , including those depicted here , are possible , but altogether they comprise at most three independent gravitational - wave observables . the mission - concept studies ran in 20112012 by esa and nasa embrace several approaches to limiting cost . reducing mass is a broadly useful strategy , because it allows for launch on smaller , cheaper rockets , and because mass has been shown to be a good proxy for mission complexity , and therefore implementation cost . esa s ngo design envisages interferometric links along two arms rather than lisa s three , resulting in an asymmetric configuration with one full and two half spacecraft . as a consequence , only one tdi observable can be formed , eliminating the capability of measuring two combinations of gw polarizations simultaneously . propellant may be saved by placing the spacecraft closer together ( with arms 13 mkm rather than 5 mkm ) and closer to earth . at low frequencies , the effect of shorter armlengths is to reduce the response to gws proportionally ; for the same test - mass noise , sensitivity then decreases by the same ratio . at high frequencies , the laser power available for position measurement increases as l , since beams are broadly defocused at millions of kms , improving shot noise , but not other optical noises , by a factor l ( in rms ) . as a consequence , the sweet spot of the lisa sensitivity shifts to higher frequencies , although one may instead plan for a less powerful laser , for further cost savings . spacecraft orbits that are different than lisa s , such as geocentric options , or flat configurations that lie in the ecliptic plane would also alter gw - signal modulations , and therefore parameter - estimation performance . reducing mission duration also saves money , because it reduces the cost of supporting the mission from the ground , and it allows for shorter warranties on the various subsystems . conversely , missions are made cheaper by accepting more risk of failure or underperformance , since risk is retired by extensive testing and by introducing component redundancy , both of which are expensive . the nasa study also explored replacing lisa s custom subsystems with variants already flown on other missions ( as in omega ) , or eliminating some of them altogether ( as in lagrange ) . however , the significant performance hit and additional risk incurred by such steps is not matched by correspondingly major savings , because the main cost driver for lisa - like missions is the necessity of launching and flying three ( or more ) independent spacecraft . switching to atom interferometry would make for very different mission architectures , but the nasa study finds that an atom - interferometer mission would face many of the same cost - driving constraints as a laser - interferometer mission [ 215 , 43 ] . indeed , the overarching conclusion of the nasa study is that no technology can provide dramatic cost reductions , and that scientific performance decreases far more rapidly than cost . thus , staying the course and pursuing a modestly descoped lisa - like design , whenever programs and budgets will allow it , may yet be the most promising strategy for gw detection in space . the deci - hertz interferometer gravitational wave observatory ( decigo [ 408 , 256 , 257 ] ) is a proposed japanese mission that would observe gws at frequencies between 1 mhz and 100 hz , reaching its best ( h 10 ) sensitivity between 0.1 and 10 hz , and thus bridging the gap between lisa - like and ground - based detectors . prior to decigo , the possibility of observing gws in the decihertz band had been studied in the context of a possible follow - up to lisa , the big bang observer ( bbo [ 357 , 135 , 138 ] ) . the final decigo configuration ( 2024 + ) envisages four clusters in an earth - like solar orbit , each cluster consisting of three drag - free spacecraft in a triangle with 1000-km arms . gws are measured by operating the arms as a fabry - prot interferometer , which requires keeping the arm - lengths constant , in analogy to ground - based interferometers and in contrast to lisa s transponding scheme . decigo s test masses are 100 kg mirrors , and its lasers have 10 w power . the roadmap toward decigo includes two pathfinders : the single - spacecraft decigo pathfinder consists of a 30 cm fabry - prot cavity , and it could detect binaries of 1010 m black holes if they exist near the galaxy ; next , pre - decigo would demonstrate the decigo measurement with three spacecraft and modest optical parameters , resulting in a sensitivity 10100 times worse than one of the final decigo clusters . the decigo science objectives include measuring the gw stochastic background from standard inflation ( with sensitivity down to gr 2 10 ) , and determining the thermal history of the universe between the end of inflation and nucleosynthesis [ 325 , 274 ] ; searching for hypothesized primordial black holes ; characterizing dark energy by using neutron - star binaries as standard candles ( either with host redshifts , or by the effect of cosmic expansion on the inspiral phasing [ 408 , 334 ] ) ; illuminating the formation of massive galactic black holes by observing the coalescences of intermediate - mass ( 1010 m ) systems ; constraining the structure of neutron stars by measuring their masses ( in upwards of 100000 detections per year ) ; and even searching for planets orbiting neutron - star binaries . decigo can also test alternative theories of gravity , as reviewed in : by observing neutron - star - intermediate - mass - black - hole systems , it can constrain the dipolar radiation predicted in brans - dicke scalar - tensor theory ( see section 5.1.3 in this review ) better than lisa , and four orders of magnitude better than solar - system experiments ; it can constrain the speed of gws , parametrized as the mass of the graviton ( section 5.1.2 ) three orders of magnitude better than solar - system experiments , although not as well as lisa ; by observing binaries of neutron stars and stellar - mass black holes , it can constrain the einstein - dilaton - gauss - bonnet and dynamical chern - simons theories ; it can measure the bulk ads curvature scale that modifies the evolution of black - hole binaries in the randall - sundrum ii braneworld model ; and it can even look for extra polarization modes ( section 5.1.1 ) in the stochastic gw background . in the rest of this review we concentrate on the tests of gr that will be possible with low- , rather than mid - frequency gw observatories . nevertheless , many of the studies performed for lisa - like detectors are easily extended to the ambitious decigo program , which would probe gw sources of similar nature , but of different masses or in different phases of their evolution . the low - frequency gw band , which we define as 10 hz f 10 hz , is astrophysically appealing because it is populated by a large number of sources , which are relevant to understanding stellar evolution and stellar populations , to probing the growth and evolution massive black holes ( mbh ) and their links to galaxies , and possibly to observing the primordial universe . space - based interferometric detectors such as lisa and elisa are sensitive over this entire band . figure 2 illustrates the gw strength , as a function of gw frequency , for a variety of interesting sources classes . the lisa and elisa baseline sensitivities are overlaid , illustrating the potential reach of interferometric detectors . the area above the baseline curve is generically called discovery space ; the height of a source above the curve provides a rough estimate of the expected signal - to - noise ratio ( snr ) with which the source could be detected using matched - filtering techniques . snr and matched filtering are important concepts that underlie much of what will be discussed elsewhere in this review , so we explain them briefly in this section . figure 2the discovery space for space - based gw detectors , covering the low - frequency region of the gw spectrum , 10 hz f 0.1 hz . the discovery space is delineated by the lisa threshold sensitivity curve in black , and by the elisa sensitivity curve in red ( the curves were produced using the online sensitivity curve and source plotting website ) . this region is populated by a wealth of strong sources , often in large numbers , including mergers of mbhs , emrls of stellar - scale compact objects into mbhs , and millions of close - orbiting binary systems in the galaxy . thousands of the strongest signals from these galactic binary systems should be individually resolvable , while the combined signals of millions of them produce a stochastic background at low frequencies . these systems provide ample opportunities for astrophysical tests of gr for gravitational - field strengths that are not well characterized and studied in conventional astronomy . the discovery space for space - based gw detectors , covering the low - frequency region of the gw spectrum , 10 hz f 0.1 hz . the discovery space is delineated by the lisa threshold sensitivity curve in black , and by the elisa sensitivity curve in red ( the curves were produced using the online sensitivity curve and source plotting website ) . this region is populated by a wealth of strong sources , often in large numbers , including mergers of mbhs , emrls of stellar - scale compact objects into mbhs , and millions of close - orbiting binary systems in the galaxy . thousands of the strongest signals from these galactic binary systems should be individually resolvable , while the combined signals of millions of them produce a stochastic background at low frequencies . these systems provide ample opportunities for astrophysical tests of gr for gravitational - field strengths that are not well characterized and studied in conventional astronomy . in a particular gw search based on the evaluation of a detection , the detection snr of a gw signal is defined as the ratio of the expectation value when the signal is present to the root - mean - square average of when the signal is absent . typically the output s of the detector is modeled as the sum of a signal \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bf{h}}(\vec \theta)$\end{document } , depending on parameters \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec \theta}$\end{document } , and of instrumental noise n. it is normally assumed that the noise is stationary and gaussian , and that it has uncorrelated frequency components (f ) . under these assumptions , the statistical fluctuations of noise are completely determined by the one - sided power spectral density ( psd ) sh(f ) , which is defined by the equation 28\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left\langle { \tilde n(f)\tilde n(f^{\prime})^{\ast } } \right\rangle = { 1 \over 2}{s_h}(f)\delta ( f - f^{\prime}),$$\end{document } where denotes the expectation value over realizations of the noise ( also known as the ensemble average ) , and the asterisk denotes complex conjugation . this gives rise to a natural definition of an inner product on the space of possible waveform templates , 29\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$({{\bf{h}}_1}{\vert}{{\bf{h}}_2 } ) = 2\int\nolimits_0^\infty \left [ { { { \tilde h_1^{\ast}{{\tilde h}_2 } + { { \tilde h}_1}\tilde h_2^{\ast } } \over { { s_h}(f ) } } } \right]\,\,\,{\rm{d}}f.$$\end{document } in particular , the ( sampling ) probability of a given realization of noise n0 is just 30\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p({\bf{n } } = { { \bf{n}}_0 } ) \propto \exp \left [ { - { 1 \over 2}({{\bf{n}}_0}{\vert}{{\bf{n}}_0 } ) } \right].$$\end{document } searching the data for gw signals usually involves applying a linear filter k(t ) to compute the statistic 31\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$w = \int\nolimits_{- \infty}^\infty k(t)s(t){\rm{d}}t = \int\nolimits_{- \infty}^\infty { \tilde k^{\ast}}(f)\tilde s(f){\rm{d}}f.$$\end{document } redefining the filter by setting \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\tilde f(f ) = \tilde k(f){s_h}(f)$\end{document } yields the overlap ( f|s ) . the corresponding snr is 32\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s \over n } = { { ( f{\vert}h ) } \over { \sqrt { \left\langle { ( f{\vert}n)(f{\vert}n ) } \right\rangle } } } = { { ( f{\vert}h ) } \over { \sqrt { ( f{\vert}f)}}},$$\end{document } and from the cauchy - schwarz inequality we have 33\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\left({{s \over n } } \right)^2 } = { { { { ( f{\vert}h)}^2 } } \over { ( f{\vert}f ) } } \leq { { ( f{\vert}f)(h{\vert}h ) } \over { ( f{\vert}f ) } } = ( h{\vert}h),$$\end{document } with equality when f = h. this shows that the matched filter obtained when f = h is the optimal linear statistic to search for the signal h in noise characterized by the psd sh(f ) . furthermore , the optimal matched - filtering snr is just \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt { ( h|h)}$\end{document}. future references to snr in this review will always refer to this mathematical object . the three main types of gw sources in the low - frequency band are massive black hole ( mbh ) binaries , extreme mass - ratio inspirals ( emris ) , and galactic binaries . these systems typically form following the merger of two galaxies , when the mbhs originally in the centers of the two pre - merger galaxies reach the center of the merged galaxy and form a binary . these binaries generate very strong gw emission and can be detected by space - based gw detectors out to cosmological distances . the signals will be observed from the time they enter the detector band , at 10 hz , until the signal cuts off after the objects have merged . the systems evolve through an inspiral , as the objects orbit one another on a nearly circular orbit of gradually shrinking radius , followed by the merger , and then the ringdown . this last phase of the signal is generated as the black - hole merger product , which is initially perturbed in a highly asymmetric state , settles down to a stationary and axisymmetric configuration . during the inspiral phase the field strength is moderate and the velocity is low compared to the speed of light , so the system can be modeled using post - newtonian theory [ 289 , 477 , 263 , 478 , 84 ] . during the merger the system is highly dynamical and requires full nonlinear modeling using numerical relativity [ 367 , 102 , 42 , 398 ] . the final ringdown emission can then be computed using black - hole perturbation theory , since the perturbations of the object away from stationarity and axisymmetry are small . there have been several attempts to develop models that include all three phases of the emission in a single framework . the effective one - body ( eob ) model was initially developed analytically , and is based on the idea of modeling the dynamics of a binary by describing the relative motion of binary components as the motion of a test particle in an external spacetime metric ( the metric of the effective single body ) [ 99 , 100 , 141 ] . the eob model includes a smooth transition to plunge and merger , and then a sharp transition to ringdown . the model incorporates a number of free parameters that have now been estimated by comparison to numerical - relativity simulations [ 101 , 144 ] . ( eobnr ) model described non - spinning black holes , but the formalism has now been extended to include the effects of non - precessing spins [ 345 , 51 , 438 , 44 ] . the other model that includes all phases of the waveform is the phenomenological inspiral - merger - ringdown ( pimr ) developed by ajith et al . this model was constructed by directly fitting an ansatz for the frequency - domain waveform , which was motivated by analytic and numerical results , to the output of numerical relativity simulations . this model has also now been extended to include the effect of non - precessing spins . emris consist of stellar - mass ( 0.550 m ) compact objects , either white dwarfs , neutron stars , or black holes , that orbit mbhs . these are expected to occur in the centers of quiescent galaxies , in which a central mbh is surrounded by a cluster of stars . interactions between these stars can put compact objects onto orbits that come very close to the mbh , leading to gravitational capture . emris are not as strong gw emitters as mbh binaries , but are expected to be observable ( if sufficiently close to us ) for the final few years before the smaller object merges with the central mbh , when the emitted gws are in the most sensitive part of the frequency range of space - based detectors . emris will also undergo inspiral , merger , and ringdown , but the signals from the latter two phases are likely to be too weak to be detected . during the inspiral , emris are also expected to be on eccentric and inclined orbits , which colors the emitted gws with multiple frequency components . during an emri , the smaller object spends many thousands of orbits in the strong - field regime , where its velocity is a significant fraction of the speed of light , so post - newtonian waveforms are inapplicable . however , the extreme mass ratio means that black - hole perturbation theory can be used to compute waveforms , using the mass ratio as an expansion parameter [ 362 , 394 ] . stellar binaries in our own galaxy with two compact object components will also be sources for space - based detectors if the binary period is appropriate ( 10 10 s ) . the binary components must be compact to ensure that the binary can reach such periods without undergoing mass transfer . theoretical models and observational evidence suggest that there may be many millions of such binaries that are potential sources . these systems are not expected to evolve significantly over the typical lifetime of a space mission , and will therefore produce continuous and mostly monochromatic gw sources in the band of space - based detectors . for a small number of systems it will be possible to observe a linear drift in frequency over the observation time , due to either gw - driven inspiral or mass transfer . these systems remain in the weak - field regime through their observation , and their emission can be represented accurately using the quadrupole formula [ 353 , 354 ] . in addition to what they will teach us about astrophysics , all these sources are prospective laboratories for testing gravitation . mbh binaries are strong - field systems that yield gw signals with large snrs , making signal detection and characterization less ambiguous than for weaker sources . emris provide detailed probes of mbh spacetimes , thanks to the large number of strong - field waveform cycles that will be observable from these systems . individually - resolvable compact galactic binaries can also be used to test gr , because their waveforms and evolution are well understood and easily described using model templates . in addition , many such binaries may be detectable with both gw and em observatories , providing opportunities to test the propagation of gws relative to em signals . many approaches to constraining alternative theories are proposed as null experiments , where the assumption is that gw observations will validate the predictions of gr to the level of the detector s instrumental noise . the size of residual deviations from gr is then constrained by the size of the errors in the gw measurement . however , in order to perform these null experiments , it is necessary to have accurate models for the gws predicted in gr . the appropriate modeling scheme , assuming gr and a system in vacuum , depends on the system under consideration , as described above . additional modeling complications arise from astrophysical phenomena , since tidal coupling , extended body effects , and mass transfer can all leave an impact on source evolution , complicating the interpretation of the observed gws . the very capabilities of space - based detectors also introduce complications : in contrast to the rare appearance of gw signals in the output of ground - based interferometers , the data from space - based missions will contain the superposition of millions of individual continuous signals . we expect that we will be able to resolve individually thousands of the strongest sources . thus , we must deal with problems of confusion ( where the presence of many interfering sources complicates their individual detection ) , subtraction ( where strong signals must be carefully modeled and removed from the data before weaker sources , overlapping in frequency , become visible ) , and global fit ( where the parameters of overlapping sources have correlated errors that must be varied together in searches and parameter - estimation studies ) . these issues have not escaped the attention of lisa scientists , and have been tackled both theoretically , and in a practical program of mock data challenges [ 37 , 39 , 450 ] . all these issues in data analysis and modeling will impact the prospects for tests of gr using the observations . little work has been done to date to estimate these impacts , but we will discuss relevant studies where appropriate . in the remainder of this section we will briefly describe the three principal source classes expected in the low - frequency band . for each source type , we will discuss the astrophysics of the systems , the estimated event rates for ( e)lisa observations , their potential astrophysical implications , and their applications to testing gr , with pointers to later sections that describe the tests in more detail . we focus on the three types of source that we introduced above and that are most likely , from an astrophysical point of view , to be observed by lisa - like observatories : mbh coalescences , emris , and galactic binaries . it is possible that a space - based detector like lisa or elisa could detect other sources that could be used for tests of relativity . for instance , if intermediate - mass black holes with mass between 100m and 10m do exist , they could be observed as intermediate - mass - ratio inspirals ( imris ) when they inspiral into the mbhs in the centers of galaxies . these systems would have the potential for the same kind of tests of fundamental physics as emris , but with considerably larger snr at the same distance and hence would be observable to much greater distances . cosmological gw backgrounds might also be observed , generated by processes occurring at the tev scale in the early universe , which would provide constraints on the physics of the early universe and inflation . we refer the reader to [ 21 , 20 ] for discussions of these sources . we regard them as somewhat more speculative than the other three source types and we do not consider them further here . most ( if not all ) galactic nuclei come to harbor a mbh during their evolution [ 296 , 377 ] , and individual galaxies are expected to undergo multiple mergers over their lifetime . it follows that the formation of mbh binaries following galaxy mergers is an expected outcome . the mergers of such binaries will be among the strongest sources of low - frequency gws . the rate at which mbh binaries merge in the universe is uncertain at best , but these events will be detectable by lisa - like detectors to extremely large distances , probing an enormous volume of the visible universe . the detection of any mbh mergers , even at a low rate , would produce interesting astrophysical results . for a circular binary , the mass of a system that merges at a frequency fm is roughly 34\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_t } \approx { 10 ^ 5}{m _ \odot}\left({{{40\,\,{\rm{mhz } } } \over { { f_m } } } } \right);$$\end{document } the frequency range accessible to space - based gw detectors extends from a few 10 hz to a few 10 hz , which sets the sensitive mass range to 1010 m. mbh mergers as low - frequency gw sources . predictions for the observable population of mbh mergers are based on merger - tree structure - formation models . the overall merger rate depends on the detailed mechanisms of evolutionary growth of the mbh population . the energy budgets of active galactic nuclei suggest that mbhs could grow by efficient accretion processes , while other considerations suggest that mergers could contribute significantly to their early growth . studies of early cosmic structure indicate that mbhs form from the coalescence of many smaller seed black holes . many models based on this idea have been developed and simulated numerically [ 216 , 460 , 437 , 60 ] . the number of events detectable by lisa was estimated to be in the range 3300 per year , with a spectrum of masses in the range 1010 m. the predicted event rate is not much different for elisa , although some of the marginally detectable events involving lighter black holes at high redshift will no longer be observable . figure 3 shows contours of constant snr ( as seen by elisa ) in the redshift - total - mass plane for equal - mass , nonspinning mbh mergers ( left panel ) and in the total - mass - mass - ratio plane for mbh mergers at a fixed redshift of z = 4 ( right panel ) . for typical events at z 4 , the snrs of elisa observations are expected to exceed 100 for total masses 5 10 m. for comparablemass mergers at lower redshifts , elisa observations could exceed snrs of 1000 . snrs for lisa are typically factors of 23 higher . for total mass > 10 m , a significant fraction of this snr comes from the final merger and ringdown phase . the left - hand panel shows contours in the total - mass - redshift plane for equal - mass binaries , while the right - hand panel shows contours in the total - mass - mass - ratio plane for sources at redshift z = 4 . the left - hand panel shows contours in the total - mass - redshift plane for equal - mass binaries , while the right - hand panel shows contours in the total - mass - mass - ratio plane for sources at redshift z = 4 . image reproduced by permission from . several detection algorithms for mbh binaries have been studied by a number of different groups , encouraged in part by the mock lisa data challenges . these include markov chain monte carlo techniques , time - frequency analysis , particle - swarm optimization , nested sampling , and genetic algorithms . the most recent mock - data challenge included multiple spinning mbh binary signals in the same dataset , and multiple groups demonstrated their ability to recover these binaries from the lisa data stream . gw observations will directly probe the character of the black holes on scales comparable to the size of the event horizons . how the information about the black holes ( such as their spin and mass ) is encoded in the waveform is a topic of much focused research ( see , e.g. , [ 275 , 411 , 117 , 421 ] for reviews ) . according to gr , any astrophysical black hole is fully described by just ten numbers , encoding its mass , position , momentum , and spin . correspondingly , a binary is fully described by 20 parameters , three of which , related to its center - of - mass momentum , can not be measured from the gw signal . the precision with which gw observations will be able to extract the parameters characterizing a mbh binary system has been the subject of extensive investigation . the first comprehensive study considered the lisa determination of the sky location , luminosity distance , and mass of mbh systems , using a simple model of lisa s orbital motion around the sun . later studies considered the angular resolution of detectors in precessing - plane configurations like lisa s , as well as ecliptic - plane interferometers that lie flat in the ecliptic as they orbit the sun . more recent studies have considered more complete mbh - binary waveforms with spin effects , higher harmonics , and with merger and ringdown signals , and have looked at the effect of these corrections on lisa s parameter - estimation ability [ 455 , 29 , 442 , 365 , 33 , 266 , 307 ] . these papers indicate that observations with a lisa - like detector will be able to determine the masses of the two binary components to 0.010.1% , the spins to 0.0010.1% , the sky position of the binary to 110 deg , and the luminosity distance to 0.110% . for elisa , these parameter - estimation studies witness the quality of the information that will be available to astrophysicists for the phenomenological exploration of the astrophysical character of mbh systems . of particular interest is the evolution of black - hole spins and the final spin of merger remnants [ 80 , 242 ] , which gw observations should be able to shed some light on . in addition , the set of detected mbh merger systems will provide constraints on the formation history of mbhs [ 360 , 359 , 241 , 308 , 355 ] . in [ 405 , 198 ] it was shown that a detector like lisa would be able to tell with high confidence the difference between ten different models for the growth of structure in the universe . for many of the models , the difference would already be apparent after as little as three months of data collection . a similar differentiation among models would also be possible with elisa , and both detectors will also be able to constrain mbh binaries can also be used as standard candles to probe cosmological evolution . testing relativity using mbh mergers . mbh mergers are good laboratories for testing relativity because of the high snr expected for the signals and the correspondingly large volume within which they can be observed . this makes them good systems for constraining gw polarizations ( see section 5.1.1 ) since relatively weak alternative - polarization signals could in principle be detected . they are also excellent systems for testing gw - propagation effects , such as subluminal propagation speeds and the presence of parity violations , since these effects accumulate with distance and mbh binaries can be seen to very high redshifts ( see section 5.1.2 ) . mbh binaries are also well suited for generic tests of gr based on measuring the evolution of inspiral phasing and checking it for consistency with general - relativistic predictions , since the high snr allows a large number of phasing parameters to be measured ( see section 5.2 ) . mbh binaries are also the only systems for which the quasinormal ringdown radiation , generated as the highly - distorted post - merger black hole settles down into a quiescent state , will be detectable . although the amount of energy deposited into different ringdown modes is not well understood , the frequencies and decay rates of the ringdown modes are predicted precisely in gr from perturbation theory . the mass and spin of the final black hole can be measured from a single ringdown mode ; if multiple modes are detected , the system can be used to check for consistency with the kerr metric , and therefore provides a probe of black - hole structure . for a comprehensive review of black - hole ringdowns , a more detailed discussion of the use of ringdown radiation to probe black - hole structure may be found in section 6.3 . last , the space - based gw observation of mbh binaries offers an unprecedented opportunity to probe the fully - dynamic strong - field regime of gr by comparison of the observed merger signals to the predictions of numerical relativity . this is an area that has not yet been explored in any detail and so we will not discuss it further in this review . however , it is an important subject that should be carefully explored before a space - based gw detector finally becomes a reality . an emri is the gw - emitting inspiral of a stellar - mass compact object , either a black hole , neutron star or white dwarf , into a mbh in the center of a galaxy . a main - sequence star with mean density \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar \rho = 10\,{\rm{g c}}{{\rm{m}}^{- 3}}$\end{document } will be tidally disrupted by a black hole of mass 10 m at a distance of 25 schwarzschild radii . at such separations an extra - galactic emri system will not be generating detectable amounts of gravitational radiation in the low - frequency band . the tidal - disruption radius increases with decreasing stellar density and decreasing central - black - hole mass , and the reference values used above are at the upper end of suitable values for main - sequence stars and mhz gw sources . therefore , it is not expected that the inspiral of a main - sequence star will be a candidate for an emri , with the possible exception of such sources in the galactic center , which is sufficiently nearby that radiation from an object orbiting at several tens of schwarzschild radii might be detectable . emris occur in the dense stellar clusters that are found surrounding the black holes in the cores of galaxies , and are triggered by a variety of processes : direct capture : this is the scenario , in which two - body encounters in the stellar cluster gradually perturb the compact - object orbits , changing their angular momentum . this can put a compact object onto an orbit that passes very close to the central black hole . the orbit loses energy and angular momentum in bursts of gravitational radiation emitted near periapse , and if sufficient energy is radiated , the object can be left on an orbit that is bound to the central black hole . it will then gradually inspiral into the central black hole as it loses orbital energy and angular momentum to gw emission.tidal splitting of binaries : galactic - center stellar clusters will also contain binaries , which can similarly be displaced onto orbits that pass close to the central mbh . if this happens , the binary will typically be disrupted , with one object remaining bound to the mbh and the other becoming unbound and being flung out with high velocity . if this happens to a binary containing one or two compact objects , the captured compact object will be left on a fairly tightly bound orbit a few hundred astronomical units from the mbh , and will become an emri on a nearly circular orbit .tidal stripping of giant stars : giant stars typically have a compact core surrounded by a diffuse hydrogen envelope . if such a star passes close to a mbh , the envelope can be partially or completely stripped by the tidal interaction with the mbh . this will deposit the dense core , which is essentially a white dwarf , on a close orbit about the mbh ; the system will eventually become an emri [ 145 , 150],in - situ formation : if a mbh has a massive accretion disc , the disc can become unstable to star formation . stars formed in such a way are biased toward higher masses and will therefore tend to form black - hole remnants . these remnants will be in circular , equatorial orbits around the central mbh , and will inspiral as emris under gw emission . more details on all of these processes and further references can be found in [ 18 , 16 ] . direct capture : this is the standard scenario , in which two - body encounters in the stellar cluster gradually perturb the compact - object orbits , changing their angular momentum . this can put a compact object onto an orbit that passes very close to the central black hole . the orbit loses energy and angular momentum in bursts of gravitational radiation emitted near periapse , and if sufficient energy is radiated , the object can be left on an orbit that is bound to the central black hole . it will then gradually inspiral into the central black hole as it loses orbital energy and angular momentum to gw emission . tidal splitting of binaries : galactic - center stellar clusters will also contain binaries , which can similarly be displaced onto orbits that pass close to the central mbh . if this happens , the binary will typically be disrupted , with one object remaining bound to the mbh and the other becoming unbound and being flung out with high velocity . if this happens to a binary containing one or two compact objects , the captured compact object will be left on a fairly tightly bound orbit a few hundred astronomical units from the mbh , and will become an emri on a nearly circular orbit . tidal stripping of giant stars : giant stars typically have a compact core surrounded by a diffuse hydrogen envelope . if such a star passes close to a mbh , the envelope can be partially or completely stripped by the tidal interaction with the mbh . this will deposit the dense core , which is essentially a white dwarf , on a close orbit about the mbh ; the system will eventually become an emri [ 145 , 150 ] , in - situ formation : if a mbh has a massive accretion disc , the disc can become unstable to star formation . stars formed in such a way are biased toward higher masses and will therefore tend to form black - hole remnants . these remnants will be in circular , equatorial orbits around the central mbh , and will inspiral as emris under gw emission . . the intrinsic rate at which emris occur depends on a lot of astrophysics that is rather poorly understood . the rate is strongly influenced by several processes , including : mass segregation : stellar encounters tend on average to lead to equipartition of energy . this means that after a two - body interaction the heavier object tends to be moving slower , and the lighter object tends to be moving faster . if this occurs for two objects around a mbh , the heavier object sinks in the potential of the mbh and the lighter object moves further out . components of the stellar distribution thus become segregated according to their mass . the more massive objects , which tend to be the black holes , end up closer to the mbh and are captured preferentially [ 18 , 188 , 313].triaxiality : the centers of galaxies tend to be approximately spherically symmetric , but on larger scales galactic nuclei can be triaxial . orbits in triaxial potentials tend to be centrophilic : they have a tendency to pass close to the center . this process can funnel stars that are further away from the mbh toward the center and hence increase the inspiral rate [ 233 , 364].resonant relaxation : in the standard relaxation picture , each encounter is random and uncorrelated , so stars undergo a random walk . the process is driven by the diffusion of energy which then leads to angular - momentum transfer . however , in a stellar cluster around a mbh , each star will be on a keplerian orbit , which is a fixed ellipse in space . the orbits of two nearby stars will thus exert correlated torques on one another , which can lead to a direct resonant evolution of the angular momentum [ 375 , 376 ] . this process can lead to a much more rapid diffusion of orbits into the regime where they can become emris.the schwarzschild barrier : resonant relaxation relies on the orbits having commensurate radial and azimuthal frequencies , so they remain in fixed planes over multiple orbits . in the strong - field potential of a massive object the schwarzschild barrier refers to the boundary between orbits with and without significant precession . inside this point resonant relaxation a full description of these processes and their relative importance can be found in [ 18 , 16 ] . the range of emri rates per galaxy reported in the literature is 11000 gyr for black holes , and 105000 gyr for white dwarfs , with most recent best - guess estimates of 400 gyr / 20 gyr / 7 gyr for black holes / white dwarfs / neutron stars [ 235 , 19 ] . this means that after a two - body interaction the heavier object tends to be moving slower , and the lighter object tends to be moving faster . if this occurs for two objects around a mbh , the heavier object sinks in the potential of the mbh and the lighter object moves further out . components of the stellar distribution thus become segregated according to their mass . the more massive objects , which tend to be the black holes , end up closer to the mbh and triaxiality : the centers of galaxies tend to be approximately spherically symmetric , but on larger scales galactic nuclei can be triaxial . orbits in triaxial potentials tend to be centrophilic : they have a tendency to pass close to the center . this process can funnel stars that are further away from the mbh toward the center and hence increase the inspiral rate [ 233 , 364 ] . resonant relaxation : in the standard relaxation picture , each encounter is random and uncorrelated , so stars undergo a random walk . the process is driven by the diffusion of energy which then leads to angular - momentum transfer . however , in a stellar cluster around a mbh , each star will be on a keplerian orbit , which is a fixed ellipse in space . the orbits of two nearby stars will thus exert correlated torques on one another , which can lead to a direct resonant evolution of the angular momentum [ 375 , 376 ] . this process can lead to a much more rapid diffusion of orbits into the regime where they can become emris . the schwarzschild barrier : resonant relaxation relies on the orbits having commensurate radial and azimuthal frequencies , so they remain in fixed planes over multiple orbits . in the strong - field potential of a massive object the schwarzschild barrier refers to the boundary between orbits with and without significant precession . inside this point resonant relaxation uncertainties in the event rate come not only from the intrinsic rate of capture , but also from uncertainties in the number density of black holes of suitable mass . at present , only three objects in the range 1010 m are known from kinematic measurements . using galaxy luminosity functions , the luminosity - velocity - dispersion relation and the mbh - mass - velocity dispersion relation , one infers the number density of mbhs in the lisa mass range to be roughly constant per logarithmic mass unit , and equal to a few 10 mpc . however , when black holes merge , the asymmetric radiation of linear momentum over the final few orbits imparts linear momentum ( a merger kick ) to the remnant black hole . these kicks can be large enough to eject the black hole from its host galaxy . if these black - hole merger kicks are significant for black holes merging in the centers of galaxies , the number of black holes with m < 2 10 m may therefore be significantly depleted . emri waveforms are complex , since the orbits are expected to be both eccentric and inclined with respect to the equatorial plane of the central black hole . various algorithms have been suggested for emri detection , including hierarchical matched filtering starting with short data segments , time - frequency analysis [ 467 , 200 , 194 ] , searches with phenomenological templates , and markov chain monte carlo searches [ 429 , 35 , 130 ] . indeed , emris have proven to be the most difficult source to detect in the mock lisa data challenges , but in the third challenge round two groups correctly identified the three emris with the highest - mass central black holes . several challenging aspects remain unsolved , such as dealing with source confusion and detecting the emris with lower - mass central black holes , which are expected to dominate the data ; recent progress encourages confidence that these problems will be solved by the time a space - based detector is launched . assuming that a matched - filtering snr of 30 is required for emri detection , and using the previously - quoted emri rates per black hole , it was estimated that lisa could see several hundred events out to a redshift z 1 . the intrinsic rate uncertainties mean that the actual number could be anywhere from a few tens to a few thousands . most of the events will be black - hole inspirals into mbhs of mass 1010 m. the estimate for elisa is a few tens of events [ 197 , 20 , 21 ] , although this is based on a lower snr detection threshold of 20 . ( the lower threshold is justified by the results of the mock lisa data challenges , which successfully demonstrated detection of emri events with snr of 15 , albeit with less - than - realistic signal confusion . ) emri observations will tell us about the properties of mbhs at low redshift and about the physics of nuclear stellar clusters . each emri observation can determine many of the parameters of the system , including the mass and spin of the central black hole , to accuracies of a fraction of a percent [ 46 , 238 ] . with as few as ten emri observations , lisa would be able to constrain the slope of the mbh mass function at low redshift to 0.3 , which is the level of the current observational uncertainty . elisa would have the same capability given the same number of detections . if lisa observed as many emris as current estimates predict , the constraints on the slope would improve by an order of magnitude , although the corresponding improvement for elisa would be only a factor of two . the masses of the compact objects observed in emris will provide information about mass - segregation processes , while the overall number of events encodes information about the complicated physics that determines the event rate . finally , the eccentricities and inclinations of the observed emris will provide information on the relative efficiencies of the various channels through which emris could be formed [ 18 , 16 ] . testing relativity using emris . emris are well suited for testing relativity for several reasons : the systems are expected to be clean since the small object is likely to be a black hole with no internal structure , and the influence of any gaseous material in the spacetime is expected to be negligible ( but see section 6 ) ; the waveforms can be computed very accurately using black - hole perturbation theory [ 362 , 48 ] , allowing very small deviations from the model to be identified ; the inspiral is slow , meaning that many waveform cycles are generated in the strong - field regime , and can be used to test the spacetime structure ; and the waveform structure is complex , due to the expected eccentricity and inclination of the orbits , and to the intrinsic richness of dynamics in kerr ( or kerr - like ) spacetimes [ 18 , 154 ] . the primary fundamental - physics application of emri observations is probing black - hole structure , since the emri waveforms encode a map of the spacetime geometry close to the central black hole . this can be used to test whether the central object is indeed described by the kerr metric , as we expect , or is some other kind of object . a discrepancy could indicate an astrophysical perturbation in the system , a violation of the cosmic - censorship hypothesis or of the energy conditions of gr , or even evidence for the existence of an exotic massive compact object such as a boson star . emris can also be used for tests of gw polarization ( section 5.1.1 ) , and to constrain alternative theories of gravity through detection of modifications to the inspiral rate ( see section 5.1.3 ) . the most numerous source class in the low - frequency gw band are the ultra - compact binaries of two stellar - mass compact objects : white dwarfs , neutron stars , or black holes . numerical population synthesis [ 330 , 328 ] and theoretical studies of the highly - evolved stellar population in the galaxy [ 245 , 246 ] predict that there will be 10 ultracompact binaries generating gws in the mhz band visible to space - based detectors like lisa or elisa . ultra - compact binaries as low - frequency gw sources . for an instrument with sensitivity similar to the classic lisa design , the complete population of compact galactic binaries would separate into two distinct observational classes : confused binaries and resolvable binaries . confused binaries are those with orbital frequencies that are close enough to other binaries in the population that they can not be readily distinguished from one another in the data [ 63 , 231 , 131 ] ; their signals are overlapping and can not be disentangled from one another , unless one source happens to be much closer and stronger than the others . for lisa , the confused binaries make up a stochastic foreground signal from the galaxy , which would be present in the data for all frequencies below the confusion limit fc 3 mhz . various attempts have been made to estimate the spectrum of this foreground [ 329 , 439 , 162 , 161 , 386 , 335 ] , but the final profile would also depend on details of the algorithms used to analyze the lisa data [ 278 , 131 , 431 , 136 , 90 , 290 ] . for an instrument with a sensitivity comparable to elisa , which is approximately a factor of two worse in the relevant range , this confusion foreground would be much less prominent , and perhaps not detectable [ 20 , 21 ] . sources below fc are binaries that happen to be nearby , and so dominate the emission near their central frequency . for sources above fc , the characteristic orbital frequencies are separated by a few frequency bin widths f 1/tobs , where tobs is the length of observation . an instrument like lisa would be expected to resolve as many as twenty thousand individual binaries [ 329 , 335 ] , while for elisa the number is expected to be a few thousand [ 20 , 21 , 335 ] . among the resolvable binaries these are binaries that are expected to be resolvable in the lisa / elisa data , but have already been detected and characterized using electromagnetic telescopes . these known binaries will provide early verification that the mission is operating correctly at the nominal sensitivity . the large number of compact binaries detectable throughout the entire galaxy makes these sources an exceptional resource to address questions about the stellar evolutionary history of the galaxy and the astrophysical dynamics of strong - field interactions in compact systems . if the confusion foreground is observed , it will be modulated by detector motion , a fact that can be used to estimate the multipole moments of the spatial distribution of binaries in the galaxy [ 407 , 162 ] . a detector like lisa might also be able to detect the extra - galactic population of ultra - compact binaries , providing a valuable probe of its characteristics [ 171 , 127 , 129 ] . however , an extra - galactic population is unlikely to be observed by elisa . detections of galactic binaries will be dominated by double - white - dwarf binaries , which are scarce in the galaxy , so ( e)lisa will be in a unique position to illuminate their physics . this will shed light on their formation and evolution , including the poorly - understood phase of common - envelope evolution . this is the phase during the evolution of a binary that follows unstable mass transfer . in the frame that co - rotates with the binary , the region of space around a star where orbiting material is bound to that star is known as the roche lobe . as the orbit of a binary shrinks during inspiral , the roche lobes also shrink , and one of them can eventually become smaller than the corresponding star . at that point the outer layers of the star are no longer gravitationally bound to the star so they are lost , and this mass is transferred to the other star in the binary . the process can be stable if , when the material is transferred , the orbital radius increases , the roche lobe increases , and the star therefore no longer overflows the roche lobe . typically as the star loses mass it also becomes bigger , so stable mass transfer requires the roche lobe to increase in size more quickly than the star . mass transfer can also occur unstably if , when the material is lost , the orbit shrinks , leading to further shrinking of the roche lobe and more mass transfer ( or if the rate at which the star increases in size due to mass loss is faster than the rate at which the roche lobe increases ) . after an unstable mass - transfer event , the companion star and the core of the donor star will end up as a binary contained entirely within the material that was transferred from the donor the common envelope . subsequently , energy is transferred from the orbit to the envelope , leading to shrinking of the orbit until the objects merge or , in the case of interest for ultra - compact binaries , the envelope has been completely expelled . the physics of this phase of the evolution is very poorly understood , but it is normally characterized in terms of the amount of energy transferred between the binary and the envelope . the distribution of orbital periods and masses for ultra - compact galactic binaries will encode important clues about this process . gw observations can shed light on the fraction of mass - transferring systems ; these systems may allow coincident electromagnetic observations , making them even more precise probes of astrophysical processes such as mass transfer and tidal coupling [ 403 , 475 , 404 , 474 ] . testing relativity using ultra - compact binaries . resolvable compact binaries offer a good laboratory for testing gr because their waveforms and evolution are well understood and easily described using model templates , and because a reasonable fraction of them will be visible both to ( e)lisa and to electromagnetic telescopes . ultra - compact binaries will provide constraints on massive - graviton theories , either by comparison of gw - propagation delays to the observable electromagnetic signals from the binary , or by the dispersion of the gw as the binary slowly evolves . the ultra - compact binaries also provide laboratories for testing waveform evolution , and for gw polarization and dispersion studies , precisely because they evolve slowly and are easily described by post - newtonian analysis . almost since its inception , gr was understood to possess propagating , undulatory solutions gws , described at leading order by the celebrated quadrupole formula . it took several decades to establish firmly that these waves were real physical phenomena and not merely artifacts of gauge freedom . how would gw observations test the gr description of strong gravitational interactions , and possibly distinguish between gr and alternative theories ? to answer this question we need to take a quick detour through gw data analysis . at least for foreseeable detectors , individual gw signals will typically be immersed in overwhelming noise , and therefore will need to be dug out with techniques akin to matched filtering , which by definition can only recover signals of shapes known in advance ( the templates ) , or very similar signals . a matched - filtering search is set up by first selecting a parameterized template family ( where the parameters are the source properties relevant to gw emission ) , and then filtering the detector data through discrete samplings of the family that cover the expected ranges of source parameters . the best - fitting templates correspond to the most likely parameter values , and by studying the quality of fits across parameter space it is possible to derive posterior probability densities for the parameters . after a detection , the first - order question that we may ask is whether the best - fitting gr template is a satisfactory explanation for the measured data , or whether a large residual is left that can not be explained as instrument noise , at least within our understanding of noise statistics and systematics . ( slightly more involved tests are also possible : for instance , we may divide measured signals in sections , estimate source parameters separately for each , and verify that they agree . ) if a large residual is found , many hypotheses would be a priori more likely than a violation of gr : the fitting algorithm may have failed ; another gw signal , possibly of unexpected origin , may be present in the data ; the data may reflect a rare or poorly understood instrumental glitch ; the gw source may be subject to astrophysical effects from nearby astrophysical objects , or even from intervening gravitational lenses . having ruled out such non - fundamental explanations , the only way to quantify the evidence for or against gr is to consider it alongside an alternative model to describe the data . this alternative model could be a phenomenological one ( discussed below ) or a self - consistent calculation within an alternative theory of gravity . if the alternative theories under consideration include one or more adjustable parameters that connect them to gr ( such as bd for brans - dicke theory , see section 2.1 ) , and if those parameters can be propagated through the mathematics of source modeling and gw generation , then gr template families can be enlarged to include them , and the extra parameters can be estimated from gw observations . these extra parameters may have a more phenomenological character , as would , for instance , a putative graviton mass that would affect gw propagation , without finding direct justification in a specific theory . discussed below ( section 5.1 ) fall within this class . to test gr against unconnected theories without adjustable parameters , we would instead filter the data through separate gr and alternative - theory template families , and decide which model and theory are favored by the data using bayesian model comparison , which we now describe briefly . in complex data - analysis scenarios such as those encountered for gw detectors , the techniques of bayesian inference [ 211 , 414 ] are particularly useful for making assessments about the information content of data and for studying tests of gravitational theory , where the goal is to examine the hypothesis that the data might be described by some theory other than gr . in a traditional frequentist analysis of data , one computes the value of a statistic and then accepts or rejects a hypothesis about the data ( e.g. , that it contains a gw signal ) based on whether or not the statistic exceeds a threshold . the threshold is set on the basis of a false - alarm rate , which is a statement about how the statistic would be distributed if the experiment was repeated many times . evaluating the distribution of the statistic relies on a detailed and reliable understanding of the measurement process ( noise , instrument response , astrophysical uncertainties , etc . ) . by contrast , bayesian inference attempts to infer as much as possible about a particular set of data that has been observed , instead of making a statement about what would happen if the experiment were repeated . bayesian inference relies on the application of bayes 1763 theorem : given the observed data d and a parameterized model \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m(\vec \theta)$\end{document } , the theorem relates the posterior probability of the parameters given the data , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p(\vec \theta |d , m)$\end{document } to the likelihood \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p(d|\vec \theta , m)$\end{document } of observing the data d given the parameters \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec \theta}$\end{document } , and the prior probability \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p(\vec \theta / m)$\end{document } that the parameters would take that value : 35\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p(\vec \theta { \vert}d , m ) = { { p(d{\vert}\vec \theta , m)p(\vec \theta { \vert}m ) } \over { p(d{\vert}m)}}.$$\end{document } the term \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p(d|m ) = \int { p(d|\vec \theta , m)p(\vec \theta |m)d\vec \theta}$\end{document } in the denominator is the evidence for the model m. while eq . ( 35 ) follows trivially from the definition of conditional probability , its power comes from the idea of updating the prior knowledge of a system given the results of observations . however , its practical application is complicated by the necessity of attributing priors , and the correct evaluation of likelihoods relies on the same detailed understanding of the statistical properties of the measurement noise as in the frequentist case . the evidence represents a measure of the consistency of the observed data with the model m , and can be used to compare two models ( e.g. , the gr and modified - gravity descriptions of a gw - emitting system ) by evaluating the odds ratio for model 1 over model 2 , 36\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${{\mathcal o}_{12 } } = { { p(d{\vert}{m_1})p({m_1 } ) } \over { p(d{\vert}{m_2})p({m_2})}},$$\end{document } where p(m1 ) and p(m2 ) are the prior probabilities assigned for model 1 and 2 respectively . either model would be preferred if the odds ratio is sufficiently large / small , but the decision on which hypothesis is best supported by the data is influenced by the choice of the priors , which will reflect the analyst s assessment of the relative correctness of the alternatives ( see for a discussion of this point ) . in the absence of well - defined alternative - theory foils , it may be desirable to proceed along the lines of the ppn formalism ( section 2.1 ) and immerse the gr predictions in expanded waveform families , designed to isolate differences in the resulting gw phenomenology ( section 5.2 ) . proposals to do so include schemes where the waveform - phasing post - newtonian coefficients , which are normally deterministic functions of a smaller number of source parameters , are estimated individually from the data [ 28 , 27 ] ; the ambitiously - named parameterized post - einstein ( ppe ) framework ; and the parameterization of feynman diagrams for nonlinear graviton interactions . in section 5.3 we discuss ideas ( so far rather sparse ) to use the gws from binary mergers - ringdowns to test gr . emission of gws from the bulk motion of matter ( already emphasized above ) is contrasted to processes such as mass transfer , dynamical equation - of - state effects , magnetic fields , and so on . an even stronger notion of cleanness is important for the purpose of testing gr : for the best sources , the waveform signatures of alternative theories can not be reproduced by changing the astrophysical parameters of the system this orthogonality is quantified by the fitting factor between the gr and alternative - theory waveform families . fundamental bias arises from the assumption that the underlying theory in the analysis , generally taken to be gr , is the correct fundamental description for the physics being observed , which will impact the estimation of astrophysical quantities [ 497 , 453 ] . second , many of the results presented in this section rely on the fisher - matrix formalism for evaluating the expected parameter - estimation accuracy of gw observations . as described at the beginning of section 4 , the output of a gw detector is normally modeled as a linear combination of a signal , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bf{h}}(\vec \theta)$\end{document } , and noise \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bf{n}},\,{\bf{d } } = { \bf{h}}(\vec \theta ) + { \bf{n}}$\end{document}. if the detector noise is assumed to be gaussian and stationary , the probability p(n = n0 ) is given by eq . the likelihood \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p({\bf{d}}{\rm{|}}\vec \theta { \rm{,m)}}$\end{document } is just the probability that the noise takes the value \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bf{n } } = { \bf{d } } - { \bf{h}}(\vec \theta)$\end{document } , which is 37\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p({\bf{d}}{\vert}\vec \theta , m ) \propto \exp \left [ { - { 1 \over 2}\left({{\bf{d } } - { \bf{h}}(\vec \theta)\left\vert { { \bf{d } } - { \bf{h } } } \right.(\vec \theta ) } \right ) } \right],$$\end{document } where ( a|b ) is the inner product defined in eq . ( 29 ) . writing \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\bf{d } } = h({{\vec \theta}_0})$\end{document } and assuming that we are close to the true parameters \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\vec \theta}_0}$\end{document } , so that we can use the linear approximation \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$h(\vec \theta ) = h({{\vec \theta}_0 } ) + { \partial _ j}h\delta { \theta ^j } + \cdots$\end{document } , we find that at quadratic order in j 38\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$p({\bf{d}}{\vert}\vec \theta , m ) \propto \exp \left [ { - { 1 \over 2}{\gamma _ { jk}}\left({\delta { \theta ^j } - { { ( { \gamma ^{- 1}})}^{jl}}({\bf{n}}{\vert}{\partial _ l}h ) } \right)\,\,\left({\delta { \theta ^k } - { { ( { \gamma ^{- 1}})}^{km}}({\bf{n}}{\vert}{\partial _ m}h ) } \right ) } \right],$$\end{document } where 39\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\gamma _ { ij } } = \left({\left . { { { \partial h } \over { \partial { \theta ^i } } } } \right\vert{{\partial h } \over { \partial { \theta ^j } } } } \right)$$\end{document } is the fisher information matrix . thus , to leading order the shape of the likelihood in the vicinity of its maximum is that of a multivariate gaussian with covariance matrix ( ) ( independent of n ) , and the variance of the one - dimensional marginalized posterior probability density of parameter is approximately ( )ii ( no sum over i ) . this will be achieved in the limit of high signal - to - noise ratio where the errors are small and the linear approximation is valid . the fisher matrix arises also as the cramr - rao lower bound on the variance of an unbiased estimator of the waveform parameters \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\vec \theta}$\end{document}. a full discussion of the various routes to the fisher - matrix formula and its applications may be found in . as emphasized by one of us , because the fisher matrix is built with the first derivatives of waveforms with respect to source parameters , it can only know about the close neighborhood of the true source parameters . if the estimated errors take the waveform outside that neighborhood , then the formalism is simply inconsistent and unreliable . safer , but the meaning of high is problem dependent , depending on the number of parameters that need to be estimated , on their correlation , and on the strength of their effects on the waveforms . in practice , only by carrying out a full computation of the posterior probability using , for example , monte carlo methods will it be known if the fisher matrix is providing a good guide to the shape of the posterior . however , the fisher matrix is generally much easier to compute than the full posterior , so it is widely used as a guide to the precision with which parameters of the model can be determined . in the context of testing gr , the fisher matrix can be evaluated for an expanded waveform model that includes non - gr - correction parameters , but at a set of parameters that correspond to gr . the estimated error in the correction parameter , ( )ii , can then be interpreted as the minimal size of a correction that would be detectable with a gw observation . as will points out [ 469 , ch . 10 ] , virtually any lorentz - invariant metric theory of gravity must predict gravitational radiation , but alternative theories will differ in its properties . will identifies three main properties that can be measured with gw detectors . these are the polarization , speed , and emission multipolarity ( monopole , dipole , quadrupole , etc . ) of gws in gr . in this paper , we broaden the scope of the third to include changes to the loss of energy to gws in inspiraling systems . in analogy to the three classic tests of gr ( the perihelion of mercury , deflection of light , and gravitational redshift ) we like to refer to the verification that these properties have the predicted gr values , rather than the values predicted by alternative theories , as the classic tests of gr using gws . just as ppn tests probe weak - field , slow - motion dynamics , these tests can be seen as probing the weak - field far zone , where waves have propagated far from their sources . however , the multipolarity of gws at emission and the energy that they carry away can be influenced by strong - field properties in the near zone where waves are generated . gr predicts the existence of two transverse quadrupolar polarization modes for gws ( also described as spin-2 and tensor using the language of group theory ) , usually labeled h+ and h. alternative metric theories of gravity predict as many as six polarizations ( three transverse and three longitudinal ) , corresponding to the independent electric - type components of the riemann curvature tensor , r0i0j . schematically , these components are measured by gw detectors by monitoring the geodesic deviation of nearby reference masses . the effect of different polarization modes is best illustrated by the induced motion of a ring of test particles , as in figure 4 . the response of a standard right - angle interferometer to a scalar wave is maximal when the wave propagates along one arm ; by contrast , tensor modes elicit maximal response when the wave propagates in a direction perpendicular to the plane of the detector . figure 4effect of the six possible gw polarization modes on a ring of test particles . the gw propagates in the z - direction for the upper three transverse modes , and in the x - direction for the lower three longitudinal modes . only modes ( a ) and ( b ) are possible in gr . the gw propagates in the z - direction for the upper three transverse modes , and in the x - direction for the lower three longitudinal modes . only modes ( a ) and ( b ) are possible in gr . image reproduced by permission from . direct detection . the use of gw polarization modes to test gr the sensitivity of resonant and interferometric detectors , as well as doppler - tracking and pulsar - timing measurements , to the extra modes was considered in several studies [ 343 , 227 , 412 , 461 , 292 , 300 , 324 , 280 , 331 , 15 , 118 , 89 , 225 ] . in the most general setting , the problem of disentangling the modes has eight unknowns the time series for the six polarizations , plus two direction angles that affect the projection of the modes on the detector but only six observables , corresponding to the r0i0j components . thus , the problem is indeterminate , unless the source position is known from other observations ( such as time - of - flight delays for a long - baseline network of detectors ) , or unless we restrict gws to transverse modes on theoretical grounds . space - based observatories similar to lisa have either a single independent interferometric observable or three ( if laser links are maintained across all three arms ) . thus , a detector with three active arms could in principle discriminate a non - gr polarization mode if the direction to the source is known , or if it can be determined from the measured signal ( by means of the modulations produced by orbital motion , or by triangulation between the signals measured at the three spacecraft ) . the lisa sensitivity to alternative polarization modes was assessed in , using the full tdi response ( see section 3.1 ) . at frequencies larger than the inverse light - travel time along the arms , lisa would be ten times more sensitive to scalar - longitudinal and vector modes [ ( d ) to ( f ) in figure 4 ] than to tensor and scalar - transverse modes [ ( a ) to ( c ) in figure 4 ] , because longitudinal effects can accumulate as the lasers travel between the spacecraft . at lower frequencies , these results have not yet been used to work out the constraints that lisa could place on specific alternative theories using different types of sources . in , a generic model for a system emitting dipole radiation in addition to quadrupole radiation was constructed . the model was similar in structure to the ppe models which will be discussed in section 5.2.2 . this model included both the dipolar component of the waveform , at the orbital frequency , and modifications to the gravitational wave phasing of both the quadrupole and dipole waveform components that arise from the additional energy lost into the dipole mode . in , the model was used to determine the constraints on dipole radiation emission that would be possible using ground - based gw detectors . this demonstrated that elisa would be able to place bounds on the parameter , that describes the observed amplitude of the dipole radiation relative to the quadrupole , of 10 , and bounds on the parameter , which describes the amount of binary orbital energy lost into the dipole radiation , of 10 . the parameter affects the phase evolution and so stronger bounds would be obtained for less massive systems , for which more waveform cycles will be observed in band . these bounds are , in both cases , comparable to those from observations with the einstein telescope , one order of magnitude better than those possible with advanced ligo and one order of magnitude worse than what would be possible with lisa . finn observed that solar oscillations with 5- to 10-minute periods produce gravitational strains 10 at earth , possibly within reach of space - based detectors . the detectors would measure the sun s dynamical gravitational field in the transition region where it is turning into radiation . finn showed that the field develops a significant phase shift relative to solar oscillations , which depends on the gw polarizations , and which could distinguish between scalar , vector , tensor , and scalar - tensor theories of gravity . the limit placed by such observations on the brans - dicke parameter would be weaker than current bounds from solar - system tests ; on the other hand , measuring incipient gws in the transition zone makes this a novel and possibly unique test . however , we note that finn s early exploration predates our full understanding of the design and parameters of lisa - like missions , which are likely to be less sensitive to this signal . this problem was revisited in , in which the authors assessed the sensitivity of lisa to the quadrupole ( l = 2 ) low - order normal modes ( p , f and g - modes ) of the sun . they estimated that the energy in these modes would have to exceed 10 ergs to allow a lisa detection , and that the required mode energy would be even higher for elisa . galactic binaries . among the compact galactic binaries that would be detected by a lisa - like detector , several have orbital inclinations known from optical observations . for these systems we can compute the specific linear combination(s ) of polarizations that would be appear in the data , which can then be checked for consistency . a single inconsistent binary may indicate an error in the determination of inclination or distance , but systematically inconsistent sources would hint at large non - tensor gw components . however , from general arguments the measurement accuracy for polarization amplitudes is 1/snr ( with snr a few tens at most for galactic binaries ) , so only very large corrections to gr would be detectable in this way . in gr , gravitational radiation propagates at the speed of light : vg = c. the experimental validation of this prediction can be posed as a bound on the graviton mass mg , which is exactly zero in gr ( see [ 59 , 209 ] for a broader context ) . however , it may be advisable to consider mg as a purely phenomenological parameter , since certain massive - graviton theories do not recover gr predictions such as light bending , as discussed in section 2.2.5 . weak - field measurements in the solar system already provide bounds on mg on the basis of the massive - graviton yukawa correction to the newtonian potential : 40\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$v(r ) = { m \over r}\exp ( - r/{\lambda _ g}),$$\end{document } where g = h / mg is the compton wavelength of the graviton . the corresponding gw propagation speed would be given by 41\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\left({{{{v_g } } \over c } } \right)^2 } = 1 - { \left({{c \over { { f_g}{\lambda _ g } } } } \right)^2},$$\end{document } with fg the frequency of radiation . the best solar - system tests provide the bound g > 2.8 10 km ( or mg < 4.4 10 ev ) . by contrast , binary - pulsar dynamics only provide a bound mg < 7.6 10 ev . as we discuss in this section , observations of binary gws with lisa - like detectors could provide bounds competitive with these results , with the advantage of examining a rather different sector of gravitational physics , wave propagation . two distinct methods have been proposed for this . comparing the phase of gw and em signals . this technique offers a direct comparison of the speed of gws with the speed of a radiation assumed to be null ( light itself ) . for the technique to work , sources must be observable in both light and gws , and the astrophysical delays ( if any ) between the two signals must be well understood and modeled . let the difference between the arrival times of gws and em signals be 42\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta t = \delta { t_a } - ( 1 + z)\delta { t_e},$$\end{document } where ta arises from propagation ( the very effect we wish to measure ) , and ta from different emission mechanisms or geometries . here z dlh0/c is the redshift of the source , with the luminosity distance and h0 the value of the hubble parameter . in terms of vg , 43\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\epsilon _ g } = 1 - { { { v_g } } \over c } = 1 \times { 10^{- 11}}\left({{{1\,\,{\rm{kpc } } } \over { { d_l } } } } \right)\left({{{\delta { t_a } } \over { 1\,\,{\rm{s } } } } } \right),$$\end{document } which we relate to mg using the total relativistic energy , 44\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\epsilon _ g } \equiv 1 - { { { v_g } } \over c } = { 1 \over 2}{\left({{{{m_g}{c^2 } } \over { h{f_g } } } } \right)^2},$$\end{document } where fg is the gw frequency . the measurement of t has been considered repeatedly in the literature [ 279 , 139 , 128 ] . the main difficulty lies with modeling the emission delay te : consider for instance am cvn binaries , where a low - mass helium donor has expanded to fill its roche lobe and is spilling mass onto a white - dwarf primary . the em signal from these systems is greatly affected by the light emitted from the overflow stream impacting the accretion disk , and the light curve oscillates as the system orbits , alternately flashing the impact point toward and away from the observer . the times of maximum emission can be taken as reference for the em phase , but how are they related to gw emission ? to evaluate this te , one may observe the compact binary at two epochs , ideally at opposite points across the earth s orbit [ 279 , 139 ] . under the assumption that te is constant , differencing the total t measured at the two epochs leaves a measure of ta alone . however , the subtraction reduces ta to what can be accumulated across the diameter of the earth s orbit , rather than across the entire distance to the binary . as a consequence , the strongest bound from known lisa verification binary would be g > 3 10 km ( mg < 4.6 10 ev ) . alternatively , one may concentrate on eclipsing compact binaries , where the light curve varies due to the mutual eclipses of the binary components , allowing the orientation geometry of the system to be precisely determined as a function of time , and yielding an accurate measure of te . in this case the measured ta is accumulated over the entire distance to the source . only one eclipsing binary that would be observable with lisa - like detectors is currently known , but an analysis of their statistically - expected population suggests that lisa would obtain a bound g > 2 10 km ( mg < 6 10ev ) . the reader may question whether it is appropriate to compare gravitons to photons , when the current bound on the putative mass of the photon is as high as m < 2 10 ev . however , the much higher frequency of optical photons compared to low - frequency gravitons leads to < 3 10 , much smaller than g < 10 ( for solar - system tests ) , so a comparison based on speeds is indeed appropriate . a related test using pulsar - timing observations would compare the gw - induced phase delays accumulated by photons traveling to earth from different pulsars : the delays depend on the graviton speed through a geometric factor that alters the expected hellings - downs correlation that gws will produce in the timing of pulsars located at different positions on the sky . it might also be possible to observe simultaneous em and gw signals from mbh mergers , using the approximate position of the source known from pre - merger gw observations to guide a follow - up campaign in the em spectrum . however , the nature of possible em counterparts is extremely uncertain , so differences between the gw and em phasing could be explained by uncertainties in the modeling of the em signal . therefore , it is unlikely that constraints from these systems will be competitive with galactic - binary constraints , or with the constraints from gw dispersion discussed in the following subsection . measuring the dispersion of gravitational - wave chirps . the chirping signals emitted by inspiraling binaries contain a range of frequency components : if the graviton has mass , the components propagate at different speeds , again given by eq . this effect can be modeled in the templates used to search for binary signals by including a g dependence in the waveform phasing . in the frequency - domain representation term (cf ) , where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\beta = { \pi ^2}d{{\mathcal m}_c}/\lambda _ g^2/(1 + z)$\end{document } with c the binary chirp mass , f the gw frequency , d the source distance , and z the source redshift . by comparison , the leading - order term in the post - newtonian expansion of the phasing is 3/128(cf ) , while the g correction contributes the same power of orbital frequency as the 1pn term . for space - based detectors , the best chirp - dispersion bounds will come from massive - black - hole systems ; they improve slightly with the total binary mass and with better low - frequency ( 10 10 hz ) sensitivity . however , the expected bounds depend strongly on which other physical effects ( such as spin - induced precessions , orbital eccentricity , higher waveform harmonics , the merger - ringdown phase ) will be relevant in the detected systems . as a result , a variety of predictions have appeared in the literature [ 473 , 70 , 71 , 32 , 424 , 485 , 425 , 259 , 244 ] . bounds as strong as g > a few 10 km seem possible , and would be strengthened by analyzing full catalogs of binary detections at once . instead of the chirping signals from inspiraling binaries , jones proposes a test of the gw dispersion relation using the waves from eccentric galactic binaries , which are emitted at multiple harmonics of the orbital frequencies ; if at least one galactic binary has sufficient eccentricity , jones claims sensitivity comparable to the chirp - dephasing measurements . extend the graviton - mass formalism to more general modified - gravity theories that predict violations of lorentz invariance and modified dispersion relations for gw modes , given by 45\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${e^2 } = { p^2}{c^2 } + m_g^2{c^4 } + { \mathbb a}{p^\alpha}{c^\alpha};$$\end{document } both mg and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\mathbb a$\end{document } can be constrained together , given the corresponding to specific theories , by inspiral - binary observations with ground and space - based detectors . is a conserved quantity , so left and right - circular polarized gravitational radiation propagates alike . many attempts to formulate a quantum theory of gravity require the addition of a parity - violating chern - simons ( cs ) term to the einstein - hilbert action [ 14 , 7 , 363 ] : 46\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${s_{{\rm{cs } } } } = { 1 \over { 64\pi}}\int { \theta \,r^{\ast}r{{\rm{d}}^4}x , } \quad \,\,\,{\rm{where}}\quad r^{\ast}r = { 1 \over 2}{r_{\alpha \beta \gamma \delta}}{\epsilon ^{\alpha \beta \gamma \nu}}r_{\mu \nu}^{\gamma \delta};$$\end{document } here r is the riemann tensor , is the levi - civita tensor density , and is a ( possibly ) position - dependent function that describes the coupling of the cs field to spacetime . this correction creates a difference in the propagation equations for the left- and right - circular gw polarizations , resulting in their amplitude birefringence : one circularly - polarized state is amplified through propagation , while the other is attenuated . this effect is potentially observable with lisa - like detectors for mbh - binary inspirals at cosmological distances ( see also ) , where the amplitude birefringence generates an apparent precession of the orbital plane of the binary . the cs correction accumulates with distance , and is larger for sources at higher redshifts . orbital - plane precession will also arise from general - relativistic spin - orbit coupling , but the scaling of the precession with frequency is different , so the two effects can be distinguished , at least in principle . for an equal - mass binary with redshifted masses of 10 m that is observed plane - on at a redshift z = 15 , lisa could constrain the integrated cs contribution at the level of 10 . this is several orders of magnitude better than solar - system experiments , which furthermore can only provide local constraints . thus , lisa - like detectors may provide some hints as to the very quantum nature of gravity . in theories that do not satisfy the strong equivalence principle , the internal gravitational binding energies of bodies can create a difference between the inertial dipole moment ( i.e. , the linear momentum , which is conserved ) and the gw - generating gravitational dipole moment . thus , alternative theories of gravity generally admit dipole radiation , but it is forbidden in gr , where the two moments are identical . dipole radiation would be given at leading order by 47\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h_d}\sim{1 \over { { d_l}}}{{\rm{d } } \over { { \rm{d}}t}}(m_1^g{{\bf{x}}_1 } + m_2^g{{\bf{x}}_2})\sim{{{\mu ^i}{\bf{v } } } \over { { d_l}}}\left({{{m_1^g } \over { m_1^i } } - { { m_2^g } \over { m_2^i } } } \right),$$\end{document } where x1,2 are the positions of the binary components , v their relative velocity , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m_{1,2}^i$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$m_{1,2}^g$\end{document } are their inertial and gravitational masses respectively , is the inertial reduced mass , and dl is the luminosity distance to the observer . for relativistic objects such as neutron stars ( ns ) , the gravitational binding energy can be considerable and so can be the resulting loss of energy to dipolar gws . indeed , the experimental result that the orbital decay of the binary pulsar psr1913 + 16 adhered closely to gr s quadrupole - formula prediction was sufficient to definitely falsify gr alternatives such as bimetric and stratified theories . ( amusingly , certain theories even predict that dipole radiation carries away negative energy from a binary . ) thus it is factually correct to state that the indirect detection of gws has already provided a strong test of gr . by contrast , the binary pulsar could not falsify scalar - tensor theories in this way , because these are close to gr . for instance , although dipole radiation is predicted by brans - dicke theory and changes the progression of orbital decay , the coupling parameter bd can be adjusted to approximate gr results to any desired accuracy . gr is reproduced for bd , so experimental bounds on brans - dicke are lower bounds . the hulse - taylor binary pulsar does provide a bound on bd , but one that is not competitive with solar - system tests , among which the best comes from the doppler tracking of the cassini spacecraft , which sets bd > 40000 [ 471 , 81 ] . however , other binary systems containing pulsars are known that provide constraints , which are competitive with solar - system constraints . the best constraints on scalar - tensor gravity ( and also teves gravity ) come from the pulsar - white - dwarf binary j1738 + 0333 , which provides the limit bd > 25000 . lisa - like detectors can constrain bd by looking for dipole - radiation - induced modifications in the gw phasing of binary inspirals ( monopole radiation is also present , but suppressed relative to the dipole ) , as long as at least one of the binary components is not a black hole : because of the no - hair theorem , black holes can not sustain the scalar field that would lead to a differing and m ( as was recently confirmed in full numerical - relativity simulations ) . this restriction can be circumvented by having non - asymptotically flat boundary conditions for the black hole . if the scalar field is slowly varying far from the black hole ( either as a function of time or space ) then it can support a scalar field . this scenario was investigated numerically in , which found that accelerated single black holes and black - hole binaries would emit scalar radiation , in the latter case at twice the orbital frequency . if the asymptotic scalar - field gradient that supports the black - hole scalar hair is cosmological in origin , this effect will be negligible , but the possibility does exist in general . except for these considerations , the canonical source for detecting this effect is the inspiral of a neutron star into a relatively low - mass central black hole , although the number of detections of such systems is likely to be very low . early studies [ 397 , 473 ] , based on simplified models of the waveforms and of the lisa sensitivity , estimated that for a 1.4 m neutron star inspiraling into a mbh , at fixed snr = 10 , the bd bounds would scale as 48\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\omega _ { { \rm{bd } } } } > 2 \times { 10 ^ 4}{\left({{{\mathcal s } \over { 0.3 } } } \right)^2}\left({{{100 } \over { \delta { \phi _ d } } } } \right){\left({{t \over { 1\,\,{\rm{yr } } } } } \right)^{7/8}}{\left({{{{{10}^4}{m _ \odot } } \over { { m _ \bullet } } } } \right)^{3/4}},$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal s}$\end{document } ( the sensitivity ) is a measure of the difference between the neutron - star and mbh self - gravitational binding energies per unit rest mass ; d is the dipole contribution to the gw phasing ; t is the time of observation ; and m is the mbh mass . however , this estimate is reduced by a factor of ten or more when more realistic waveforms are considered that include spin couplings [ 70 , 71 ] , spin - induced orbital precession and eccentricity . bounds can also be derived for a massive - scalar variant of brans - dicke theory , and are of order 49\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\left({{{{m_s } } \over { \sqrt { { \omega _ { { \rm{bd } } } } } } } } \right)\left({{\rho \over { 10 } } } \right ) \lesssim { 10^{- 19}}\,\,{\rm{ev,}}$$\end{document } ( where is the mass of the scalar and the detection snr ) for the intermediate - mass - ratio inspiral of a ns into a black hole with mass 10 m. these results were obtained using only the leading order correction from the scalar radiation . in the authors extended this calculation to all post - newtonian orders , but in the extreme - mass - ratio limit by using the teukolsky formalism . the conclusion , that constraints on massless scalar - tensor theories from gw observations will , in general , be weaker than those from solarsystem observations , was unchanged . the reason is that scalar - tensor theories are weak - field ( infrared ) corrections to gr and are therefore largest in the weak field , so the leading order correction captures the majority of the effect . massive scalar - tensor theories were also considered in [ 110 , 495 ] . in those theories , the primary observable consequence is the possible existence of floating orbits at which the scalar flux experiences a condition where gws scatter off the central , massive body , emerging with more energy ( extracted from the spin of the central body ) . the waves transfer that energy to the small orbiting body , increasing its orbital energy . the transition of an emri through such a floating orbit is many orders of magnitude slower than the normal emri inspiral and can last more than a hubble time . if an emri consistent with gr is observed it means that the emri not only did not pass through such a floating orbit during the timescale of the observation but could not have encountered one prior to the observation since it would not then have reached the millihertz band . therefore , an observation of a single emri can constrain the massive scalar - tensor parameter space to many orders of magnitude greater precision than current solar - system observations . a number of other suggestions have been made for low - frequency gw tests of gr that do not quite fit a modified energy - loss description . for instance , dynamical chern - simons theory introduces nonlinear modifications in the binary binding energy and dissipative corrections at the same pn order [ 426 , 483 ] that could be observed in the late inspiral , constraining the characteristic chern - simons length scale to 1010 km , comparable to current solar - system constraints ( advanced ground - based detectors could do even better , placing bounds of 10100 km ) . corrections to the inspiral phasing will also arise if the spacetime outside the central object is not described by the kerr metric or if additional energy is lost into scalar or other forms of radiation . this has been considered for various alternative theories of gravity ; we discuss these results in detail in section 6.2.6 . gw tails , which are due to the propagation of gravitational radiation on the curved background of the emitting binary , appear at a relative 1.5pn order ( c ) beyond the leading - order quadrupole radiation , and their observation would test the nonlinear nature of gr . ( this would be a null test of gr , since tails are included in the standard post - newtonian inspiral phasing ; see also the pn - coefficient tests discussed in section 5.2.1 . promoting newton s constant , g , to a function of time modifies both a binary s binding energy and gw luminosity , and therefore its phasing . a three - year observation of a 1010 m inspiral would constrain \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\dot g / g$\end{document } to 10 yr . the infinite randall - sundrum braneworld model may predict an enormous increase in the hawking radiation emitted by black holes [ 164 , 436 ] . the resulting progressive mass loss may be observed as an outspiral effect in the quasi - monochromatic radiation of galactic black - hole binaries , as a correction to the inspiral phasing of a black - hole binary and it would also affect the rate of emri events [ 306 , 484 ] . the constraints on the size of extra dimensions coming from observations with lisa will , in general , be worse than those derivable from tabletop experiments . however , decigo observations of bh - ns binary mergers would be able to place a constraint about ten times better than tabletop experiments , assuming a detection rate of 10 binaries per year . as discussed above , quantitative tests of gr against modified theories of gravity evaluate how well the measured signals are fit by alternative waveform families , or ( more commonly ) by waveform families that extend gr predictions by including one or more modified - gravity parameters , such as bd for brans - dicke theory . to set up these tests we need to work within the alternative theory to derive sufficiently accurate descriptions of source dynamics , gw emission , and gw propagation . an alternative approach is to operate directly at the level of the waveforms by introducing phenomenological corrections to gr predictions : for instance , by modifying specific coefficients , or by adding extra terms . so far these have concentrated on post - newtonian waveforms for circular , adiabatic inspirals , as described by the stationary - phase approximation in the frequency domain : 50\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde h(f ) = a{f^{- 7/6}}\exp \left [ { i\psi ( f ) + i{\pi \over 4 } } \right],$$\end{document } where f is the gw frequency ; a is the gw amplitude , given by geometrical projection factors \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal m}_c^{5/6}/{d_l}$\end{document } ( with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal m}_c } = { ( { m_1}{m_2})^{3/5}}/{({m_1 } + { m_2})^{1/5}}$\end{document } the chirp mass and dl the luminosity distance ) ; and for simplicity we omit the nontrivial response of space - based detectors , as well as the pn amplitude corrections . the phasing (f ) is expanded as 51\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\psi ( f ) = 2\pi f{t_c } + { \phi _ c } + \sum\limits_{k \in { \mathbb z } } { \left [ { { \psi _ k } + \psi _ k^{\log}\log f } \right]{f^{(k - 5)/3}}}.$$\end{document } for binaries with negligible component spins , the post - newtonian phasing coefficients k are currently known up to k = 7 ( 3.5 pn order ) , and in gr they are all functions of the two masses m1 and m2 alone ( although \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\psi _ 1 } = \psi _ { 0 - 4}^{\log } = \psi _ 7^{\log } = 0$\end{document } , and 5 is completely degenerate with c , so it is usually omitted ) [ 86 , 87 , 85 , 30 ] . propose a test of gr based on estimating all the k simultaneously from the measured waveform as if they were free parameters , in analogy to the post - keplerian formalism [ 293 , section 4.5 ] . the value and error estimated for each4 k , together with its pn functional form as a function of m1 and m2 , determines a region in the m1m2 plane . if gr is correct , all the regions must intersect near the true masses , as shown in figure 5 . the extent of the intersection provides a measure of how precisely gr is verified by a gw observation . a fisher - matrix analysis suggests that , for systems at the optimistic distance of 3 gpc , lisa could measure 0 to 0.1% and 2 and 3 to 10% , but that the fractional error on higher - order terms would be at best 1 . ( 51 ) yields differently shaped regions in the m1m2 plane , which must intersect near true mass values if gr is correct . image reproduced by permission from , copyright by aps . estimating all the binary - inspiral phasing coefficients k of eq . ( 51 ) yields differently shaped regions in the m1m2 plane , which must intersect near true mass values if gr is correct . image reproduced by permission from , copyright by aps . however , this setup may understate the power of this kind of test , since most of the estimation uncertainty in the k arises from their mutual degeneracy that is , from the fact that it is possible to vary the value of a subset of without appreciably modifying the waveform . this degeneracy should not impact the degree to which the data is deemed consistent with gr . in a follow - up paper , arun et al . propose a revised test whereby the masses are determined from 0 and 2 , while the other k(as well as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\psi _ 5^{\log}$\end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\psi _ 6^{\log}$\end{document } ) are individually estimated and checked for consistency with gr . in this case , even for sources at z = 1 ( 7 gpc ) , all the parameters can be constrained to 1% ( a few % for 4 0.1% for 3 ) , at least for favorable mass combinations . performing parameter estimation for the eigenvectors of the k fisher matrix indicates which combinations of coefficients can be tested more accurately for gr violations . however , it is not clear what significance with regards to testing gr should be ascribed to the accuracy of measuring the k , since we do not know at what level we could expect deviations to appear . by contrast , if we were to find that , say , the n regions in the m1m2 plane do not intersect , we could make the statistically - meaningful statement that gr appears to be violated at the n level . li et al . [ 284 , 285 ] propose a more satisfying formulation for these tests , based on bayesian model selection , which compares the bayesian evidence , given the observed data , for the pure - gr scenario against the alternative - gravity scenarios in which one or more of the k are modified . the issue of significance discussed above reappears in this context as the inherent arbitrariness in choosing prior probabilities for the k , but del pozzo et al . argue that this does not affect the efficacy of the model - comparison test in detecting gr violations . ( for a comprehensive discussion of model selection in the context of gw detection , rather than gr tests , see also [ 456 , 457 , 291 ] . for more recent applications of this formalism to ground - based detectors , see . ) in , yunes and pretorius propose a similar but more general approach , labeling it the parameterized post - einsteinian ( ppe ) framework . for adiabatic inspirals , they propose enhancing the stationary - phase inspiral signal with extra powers of gw frequency : 52\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${\tilde h^{{\rm{ppe}}}}(f ) = { \tilde h^{{\rm{gr}}}}(f ) \times ( 1 + \alpha { ( \pi { \mathcal m}f)^a})\exp \left [ { i\beta { { ( \pi { \mathcal m}f)}^b } } \right],$$\end{document } where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde h}^{{\rm{gr}}}}$\end{document } is given in eqs . while the initial suggestion in is to consider a , b , there are analytical arguments why a and b should be restricted to values a = /3 and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$a = \bar a/3$\end{document } , with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$b = \bar b/3$\end{document } , which reproduces arun s pn - coefficient scheme for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\bar b \ge - 5$\end{document}. nevertheless , this representation can reproduce the leading - order effects of several alternative theories of gravity ( see table 2 ) . table 2leading - order effects of alternative theories of gravity , as represented in the ppe framework [ eq . this table is copied from , except for the two entries labeled with an asterisk . the quadratic curvature ppe exponent given in was b = 1/3 , coming from the conservative dynamics . however , it was shown in that the dissipative correction is larger , giving the value b = 7/3 quoted above . the dynamical chern - simons ppe exponent given in was b = 4/3 , which was derived using the slow - rotation metric accurate to linear order in the spin . at quadratic order in the spin , the corrections to both conservative and dissipative dynamics occur at lower post - newtonian order , giving b = 1/3 . a b brans - dicke07/3 parity violating1 0variable g(t)8/3 13/3 massive graviton01 quadratic curvature07/3 * extra dimensions013/3 dynamical chern - simons+3 + 1/3 leading - order effects of alternative theories of gravity , as represented in the ppe framework [ eq . this table is copied from , except for the two entries labeled with an asterisk . the quadratic curvature ppe exponent given in was b = 1/3 , coming from the conservative dynamics . however , it was shown in that the dissipative correction is larger , giving the value b = 7/3 quoted above . the dynamical chern - simons ppe exponent given in was b = 4/3 , which was derived using the slow - rotation metric accurate to linear order in the spin . at quadratic order in the spin , the corrections to both conservative and dissipative dynamics occur at lower post - newtonian order , giving b = 1/3 . in , yunes and pretorius are motivated by the possibility of detecting gr violations , but also by the fundamental bias that would be incurred in estimating gw - source parameters using gr waveforms when modified gr is instead correct . in , cornish et al . reformulate the detection of gr violations described by ppe as a bayesian model - selection problem , similar to the pn - coefficient tests discussed in section 5.2.1 . figure 6 shows the bounds , for various fixed b , that could be set with lisa observations of m1,2 10 m binary inspirals at z = 1 and 3 . for b corresponding to modifications in higher - order pn terms ( which require strong - field , nonlinear gravity conditions to become evident ) , the bounds provided by lisa - like detectors become more competitive with respect to solar - system and binary - pulsar results ( where weak - field conditions prevail ) . figure 6constraints on phasing corrections in the ppe framework , as determined from lisa observations of 10 m massive - black - hole inspirals at z = 1 and z = 3 . the figure also includes the bounds derived from pulsar psr j0737 - 3039 , the solar - system bound on the graviton mass , and pn - coefficient bounds derived as described section 5.2.1 . the spike at b = 0 corresponds to the degeneracy between the ppe correction and the initial gw - phase parameter . constraints on phasing corrections in the ppe framework , as determined from lisa observations of 10 m massive - black - hole inspirals at z = 1 and z = 3 . the figure also includes the bounds derived from pulsar psr j0737 - 3039 , the solar - system bound on the graviton mass , and pn - coefficient bounds derived as described section 5.2.1 . the spike at b = 0 corresponds to the degeneracy between the ppe correction and the initial gw - phase parameter . ( adapted from . ) a ppe - like model including dipole radiation in addition to quadrupole radiation but no other modifications to the waveform phasing was described in and was discussed in section 5.1.1 above . the full ppe framework was extended to include all additional polarization states and higher waveform harmonics in . the final form was motivated by considering brans - dicke theory , lightman - lee theory and rosen s theory . in the most general form , eq . 52 is modified to 53\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \tilde h}^{{\rm{ppe}}}}(f ) = { { \tilde h}^{{\rm{gr}}}}(f ) \times \exp \left [ { i\beta { { ( \pi { \mathcal m}f)}^b } } \right]\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ { + ( { \alpha _ + } { f _ + } + { \alpha _ \times}{f _ \times } + { \alpha _ b}{f_b } + { \alpha _ l}{f_l } + { \alpha _ { { \rm{sn}}}}{f_{{\rm{sn } } } } + { \alpha _ { { \rm{se}}}}{f_{{\rm{se}}}})\quad \quad \quad \quad \quad \quad \quad } \\ { \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \times { { ( \pi { \mathcal m}f)}^a}\exp \left [ { - i\psi _ { { \rm{gr}}}^{(2 ) } + i\beta { { ( \pi { \mathcal m}f)}^b } } \right ] } \\ { + ( { \alpha _ + } { f _ + } + { \alpha _ \times}{f _ \times } + { \alpha _ b}{f_b } + { \alpha _ l}{f_l } + { \alpha _ { { \rm{sn}}}}{f_{{\rm{sn } } } } + { \alpha _ { { \rm{se}}}}{f_{{\rm{se}}}})\,\,\,\quad \quad \quad \quad \quad \quad } \\ { \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \times { { ( 2\pi { \mathcal m}f)}^c}{\eta ^{{1 \over 5}}}\exp \left [ { - i\psi _ { { \rm{gr}}}^{(1 ) } + i\delta { { ( 2\pi { \mathcal m}f)}^b } } \right],\,\ , } \\ \end{array}$$\end{document } in which \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\psi _ { { \rm{gr}}}^{(l)}$\end{document } is the gr phase of the lth waveform harmonic , = m1m2/(m1 + m2 ) is the symmetric mass ratio , and fa is the detector response to a gw in polarization mode a. the ppe parameters are ( { a } , { a } , a , , b , c , , d ) . the authors of considered two further variants of this scheme . one variant restricted the coefficients in the expansion so that they were not all independent , but were related to one another via energy conservation . the second variant included this interdependence of the parameters , and also accounted for modified propagation effects by introducing additional phase - difference yet , this fully extended ppe scheme has not been used to explore the constraints that will be possible with space - based detectors . an analysis using a waveform model with higher harmonics and spin precession , but not alternative polarization states , was carried out in . its authors considered modifications to a subset of the phase and amplitude parameters only , which corresponded to certain post - newtonian orders and could therefore also be interpreted in terms of modifications to the pn phase coefficients as discussed in section 5.2.1 . the estimated bounds derived using this more complete waveform model were typically one to two orders of magnitude better than previous estimates for highmass systems , but basically the same for low - mass systems . this is unsurprising , since the effects of spin - precession and higher harmonics will only be important late in the inspiral . high - mass systems generate lower frequency gws and are therefore only observable for the final stages of inspiral , merger and plunge . therefore , late - time corrections are proportionally more important for those systems . for high - mass systems , the authors of estimated that lisa would be able to measure deviations in the phasing parameters to a precision n 0.1 , 10 , 50 , 500 , 1000 for n = 1 , 0 , 1 , 3/2 , 2 respectively , where n denotes the post - newtonian order , with n the coefficient of f in the waveform phase . using the same model , they also estimated that lisa could place a bound of g > 1 10 km on the graviton compton wavelength when allowing for correlations between the different phase - modification parameters n . an extension of the ppe framework to emri systems requires a model in which orbits can be both eccentric and inclined . to develop this , vigeland et al . derive a set of near - kerr spacetime metrics that satisfy a set of conditions , including the existence of a carter - constant - like third integral of the motion , as well as asymptotic flatness . the solutions , which were previously found in , are restricted to a physically interesting subset by setting to zero any metric coefficients not required to reproduce known black - hole solutions in modified gravity , and by applying the peeling theorem ( i.e. , by requiring that the mass and spin of the black hole not be renormalized by the perturbation ) . the existence of a third integral is not a requirement for black - hole solutions , but in general its absence allows ergodic behavior in the orbits . this is discussed as a potential observable signature for deviations from gr in section 6.2.5 . however , data - analysis pipelines designed for gr waveforms may be insensitive to such qualitatively different systems . therefore the existence of a third integral is a practical assumption for interpretation once a gr - like emri has been observed . in , gair and yunes construct gravitational waveforms for emris occurring in the metrics of , based on the analytic kludge model constructed for gr emris . the waveforms provide a ppe - like model for emris that can be used in the same way as the circular ppe framework . parameter - estimation results with these ppe - emri models have not yet appeared in the literature . in , vallisneri provides a unified model - comparison performance analysis of all modified - gr tests that is valid for sufficiently - loud signals , and that yields the detection snr required for a statistically - significant detection of gr violations as a simple function of the fitting factor ff between the gr and modified - gr waveform families . the ff measures the extent to which one can reabsorb modified - gr effects by varying standard - gr parameters from their true values . vallisneri s analysis is valid in the limit of large snr , and may not be applicable to all realistic scenarios with finite snrs . an alternative to modifying frequency - domain inspiral waveforms is offered by cannella et al . they propose tests based on the effective - field - theory approach to binary dynamics , which expands the hilbert+point - mass action as a set of feynman diagrams . in this framework , gr corrections can be introduced by displacing the coefficients of interaction vertices from their gr values . for instance , multiplying the three - graviton vertex by a factor ( 1+3 ) affects the conservative dynamics of the theory in a manner similar to the ppn parameter , but also has consequences on radiation . a similar modification to the four - graviton vertex ( parameterized by 4 ) yields effects at the second post - newtonian order , so it has no analog in ppn . argue that gr - violating values of 3 and 4 would not be detectable with gw signals , but they would instead generate small systematic errors in the estimation of standard binary parameters . however , a thorough analysis of the detectability of such deviations has not been carried out , so this conclusion may be modified in the future . according to gr , black - hole mergers are the most energetically luminous events in the universe , with l 10 l 10 erg / s , regardless of mass : at their climax , they outshine the combined power output of all the stars in the visible universe . nevertheless , second - generation ground - based gw interferometers are expected to yield the first detections of black - hole mergers , but only with rather modest snrs . by contrast , lisa - like gw detectors would observe the mergers of heavier black holes , with snrs as high as hundreds or more throughout the universe , offering very accurate measurements of the merger waveforms . massive - black - hole coalescences may feature significant spins and eccentricity , further enriching the merger phenomenology [ 80 , 380 ] . the powerful merger events correspond to very relativistic velocities and very strong gravitational fields , so much that the pn expansion of the field equations can not be applied , and we must resort to very complex and costly numerical simulations . this makes it challenging to encode the effects of plausible gr modifications in the signal model . the first ppe paper makes such an attempt on the basis of a very crude model of merger - ringdown signals , which would probably be insufficient even to phase - match the gr signals themselves . broad efforts are currently under way to build phenomenological full - waveform ( inspiral - merger - ringdown ) models [ 4 , 344 , 438 ] ; these involve tunable parameters that are adjusted to match the waveforms produced by numerical relativity . such parameters could also be used to encode non - gr effects in the merger - ringdown . however , at this time designing such extensions in a principled way seems daunting . a simpler approach , proposed by hughes and menou , involves the golden binaries for which system parameters can be estimated from both inspiral and ringdown gws . the former encode the parameters of the binary , while the latter encode the parameters of the final black hole formed in the merger . the functional relation of the two sets of parameters can then be compared with the predictions of numerical relativity , providing a null test of the strong - field regime of gr . hughes and menou focus on measuring the remnant s mass deficit , which equals the total energy carried away by gws , so their definition of golden binaries selects those in which the mass deficit can be estimated to better than 5% . for lisa , these systems tend to have component masses between a few 10 m and a few 10 m , and to be found at z 23 , making up 110% of the total merger rate depending on black - hole population models . the estimates of are based on rather simple waveform models that omit a range of physical effects , so they could be seen as conservative , given that increased waveform complexity tends to improve parameter - estimation accuracy . a more complete analysis was carried out in , but in the context of ground - based gw detectors rather than space - based detectors . it has become apparent over the last few decades that the centers of most galaxies harbor massive , dark compact objects with masses in the range 1010 m. it is clear that these objects play a very important role in the evolution of galaxies . this is exemplified by the very tight measured correlation ( the m- relation ) between the mass of the central dark objects and the velocity dispersion of stars in the central spheroid [ 174 , 441 ] . it is generally accepted that the central dark objects are black holes described by the kerr metric , but there is presently no definitive proof of that assumption . the alternatives to the black - hole interpretation include dense star clusters , supermassive stars , magnetoids , boson stars , and fermion balls . support for the blackhole interpretation has arisen as a result of both observational and theoretical work . a short review of the evidence may be found in , although we summarize some key details in section 6.1 . as described in section 2.3 , the theoretical basis for the belief that these objects are kerr black holes has arisen from proofs that singularities inevitably form during gravitational collapse [ 341 , 351 , 224 ] and that the kerr solution is the unique stationary and axisymmetric black - hole solution in gr [ 248 , 114 , 379 ] . the uniqueness of the kerr solution is sometimes referred to as the no - hair theorem , since the solution is characterized by just two parameters , the black - hole mass m and angular momentum ( per unit mass ) a. the field of any vacuum , axisymmetric spacetime in gr can be characterized by a sequence of mass and current multipole moments , which we denote as ml ; and sl ; respectively [ 204 , 217 ] . for kerr spacetimes these multipole moments are all determined by the mass and spin via 54\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_l } + { \rm{i}}{s_l } = m{({\rm{i}}a)^l},$$\end{document } so these spacetimes require no additional independent parameters or hair . the proof of the uniqueness theorem relies on various assumptions beyond the validity of the einstein equations , so a demonstration of the non - kerr nature of astrophysical black holes could reveal exotic physics within gr . it might also indicate the presence of material in the spacetime outside the black - hole horizon , or a deviation from gr in the true theory of gravity . in this section we discuss the potential of space - based low - frequency gw detectors to probe the structure of massive compact objects and the possible interpretation of these results . short reviews of the prospects for testing relativity with measurements of black - hole hair can be found in [ 73 , 191 ] . the observational evidence for the presence of black holes in the centers of galaxies has come mainly from the studies of active galactic nuclei ( agn ) . these are known to be extremely energetic and also compact typical luminosities of 10 erg s are produced in regions less than 10 m across . the inferred agn efficiency of 10% is much greater than the typical efficiencies of nuclear fusion processes 1% ) , implying the need for a very deep relativistic potential . x - ray observations show variability on timescales of less than an hour , while observations of iron lines indicate the presence of gas moving at speeds of several thousand km per second . radio observations of water maser discs are consistent with keplerian motion around very compact central objects . in the spiral galaxy ngc 4258 vlba observations have indicated a disc with an inner ( detected ) edge at 0.1 pc , around an object of mass 3.6 10 m . in addition , about 10% of agns are associated with jets , which move at highly relativistic velocities and persist for millions of years . this requires a relativistic potential that has a preferred axis that is stable over very long timescales . agns are also remarkably similar over several decades of mass , which favors the black - hole explanation , again because kerr black holes are characterized by just m and a. in the milky way , evidence for the presence of a black hole coincident with the sgr a * radio source has come from observations of stellar motions . these are completely consistent with keplerian motion around a point source of mass m 4 10 m [ 206 , 205 ] . one star , s2 , has been observed for one complete orbit and from this it has been possible to put a limit of 0.066 on the extended fraction of the central mass that could be contained between pericenter and apocenter of the orbit of s2 . at perihelion s2 was 100 au from the central object , which provides a fairly tight constraint on its compactness . electromagnetic observations can rule out stellar clusters as the explanation for the massive central objects , but some of the exotic alternatives remain . x - ray emission comes from the very inner regions of accretion discs , but uncertainties in the radius from which the emission is coming and in the mass and spin of the central object limit their utility for probing the structure of the central object . it is also possible to construct very compact boson star spacetimes that could not be ruled out from electromagnetic observations alone . by contrast , gw observations will probe the spacetime structure as the object proceeds through the inspiral and then passes the innermost stable orbit and plunges into the horizon of the central object , if a horizon exists . we discuss the prospects for using gw observations to probe black - hole structure in the following sections 6.2 and 6.3 . equation ( 54 ) tells us that a kerr black hole is uniquely characterized by two parameters . if we can measure three multipole moments of the spacetime , we can check that they are consistent with eq . if they are not , then the object can not be a kerr black hole . boson stars will typically have more independent multipole moments . in a certain class of models of rotating boson stars , the boson star can be uniquely characterized by three multipole moments [ 389 , 73 ] , so a lisa measurement of four multipole moments could also exclude these models as an explanation of the data . gws from emris are complicated superpositions of different frequency components , found at harmonics of the three fundamental frequencies of the orbit : the orbital frequency and the frequencies of precession of the perihelion and of the orbital plane . this complex structure encodes detailed information about the spacetime in which the gws are generated . if the spacetime is assumed to be stationary and axisymmetric , it can be shown that the einstein equations reduce to a single equation , the ernst equation , for a complex scalar function , the ernst potential . by using the ernst potential and expressions due to fodor et al . that relate this potential to the multipole moments of the spacetime , ryan was able to study the properties of orbits in vacuum and axisymmetric spacetimes that possess an arbitrary set of mass and current multipole moments . however , if such an orbit is given a small radial or vertical perturbation , it will undergo small oscillations at frequencies ( the epicyclic frequencies ) that correspond to the perihelion or orbital plane precession frequencies of nearly circular and nearly equatorial orbits respectively . these frequencies can be readily computed . for the arbitrary stationary axisymmetric spacetimes considered in one finds 55\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { { \omega _ r } } \over \omega } = 3{{(3m\omega)}^{{2 \over 3 } } } - 4{{{s_1 } } \over { { m^2}}}(m\omega ) + \left({{9 \over 2 } - { 3 \over 2}{{{m_2 } } \over { { m^3 } } } } \right){{(m\omega)}^{{4 \over 3 } } } - 10{{{s_1 } } \over { { m^2}}}{{(m\omega)}^{{5 \over 3}}}\quad \quad \quad \quad \quad \quad \quad } \\ { + \left({{{27 } \over 2 } - 2{{s_1 ^ 2 } \over { { m^4 } } } - { { 21 } \over 2}{{{m_2 } } \over { { m^3 } } } } \right){{(m\omega)}^2 } + \left({- 48{{{s_1 } } \over { { m^2 } } } - 5{{{s_1}{m_2 } } \over { { m^5 } } } + 9{{{s_3 } } \over { { m^4 } } } } \right){{(m\omega)}^{{7 \over 3 } } } + \cdots , } \\ { { { { \omega _ z } } \over \omega } = 2{{{s_1 } } \over { { m^2}}}(m\omega ) + { 3 \over 2}{{{m_2 } } \over { { m^3}}}{{(m\omega)}^{{4 \over 3 } } } + \left({7{{s_1 ^ 2 } \over { { m^4 } } } + 3{{{m_2 } } \over { { m^3 } } } } \right){{(m\omega)}^2}\quad \quad \quad \quad \quad \quad \quad \quad \quad \quad \quad } \\ \end{array}$$\end{document } 56\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$+ \left({11{{{s_1}{m_2 } } \over { { m^5 } } } - 6{{{s_3 } } \over { { m^4 } } } } \right){(m\omega)^{{7 \over 3 } } } + \cdots , $ $ \end{document } where is the angular ( ) frequency of the circular orbit being perturbed , r and z are the perihelion and orbital plane precession frequencies , and ml / sl denote the mass / current multipole moments of the spacetime metric , as in eq . ( 54 ) . the primary conclusion from eqs . ( 55)(56 ) is that the various multipole moments enter the different terms in this expansion at different orders in . the precession frequencies and orbital frequency could be extracted from gw observations , so these expansions are , in principle , observable . we can use this information to map the spacetime structure near the central object and verify that the multipole moments are consistent with the no - hair property ( 54 ) that we expect for a kerr black hole . bothrodesy or holiodesy5 by analogy with geodesy , in which observations of the motion of satellites are used to probe the gravitational field of the earth . the multipole moments are also encoded in the total orbital energy lost as the orbital frequency changes by a unit logarithmic interval 57\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\begin{array}{*{20}c } { { { \delta e(f ) } \over \mu } = { 1 \over 3}{{(m\omega)}^{{2 \over 3 } } } - { 1 \over 2}{{(m\omega)}^{{4 \over 3 } } } + { { 20 } \over 9}{{{s_1 } } \over { { m^2}}}{{(m\omega)}^{{5 \over 3 } } } + \left({- { { 27 } \over 8 } + { { { m_2 } } \over { { m^3 } } } } \right){{(m\omega)}^2 } } \\ { { { 28 } \over 3}{{{s_1 } } \over { { m^2}}}{{(m\omega)}^{{7 \over 3 } } } + \left({- { { 225 } \over { 16 } } + { { 80 } \over { 27}}{{s_1 ^ 2 } \over { { m^4 } } } + { { 70 } \over 9}{{{m_2 } } \over { { m^3 } } } } \right){{(m\omega)}^{{8 \over 3 } } } } \\ { + \left({{{81 } \over 2}{{{s_1 } } \over { { m^2 } } } + 6{{{s_1}{m_2 } } \over { { m^5 } } } - 6{{{s_3 } } \over { { m^4 } } } } \right){{(m\omega)}^3 } + \cdots } \\ \end{array}$$\end{document } a more powerful observable than the three discussed so far is the number of cycles that a trajectory spends near a particular frequency 58\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\delta { \mathcal n}(f ) = { { { f^2 } } \over { { \rm{d}}f/{\rm{d}}t } } = { f^2}{{{\rm{d}}e/{\rm{d}}f } \over { { \rm{d}}e/{\rm{d}}t}},$$\end{document } but this is not as clean an observable as the precession frequencies , since it requires computing the rate of energy loss to gws in an arbitrary spacetime . ryan used this formalism in conjunction with a post - newtonian waveform model to estimate lisa s capability to measure the spacetime multipoles . he considered nearly circular and nearly equatorial inspirals , and found that lisa s ability to determine the spacetime multipoles degraded as more multipoles were included in the waveform model . the conclusion was that lisa would be able to make moderately accurate measurements of the lowest three multipole moments , but probably no more . table 3accuracy with which a lisa observation could determine the multipole moments of a spacetime decreases as more multipoles are included in the model , taken from ryan . results are shown for two typical cases , a 10 m + 10 m inspiral and a 10 m + 10 m inspiral ; in both cases the snr of the inspiral is 10./m m / m l max log10(m / m)log10(s1)log10(m2)log10(s3)log10(m4)13.73.5101023.02.91.832.31.91.30.741.51.31.10.11.013.32.8101022.51.00.331.20.10.80.941.00.10.81.21.8 accuracy with which a lisa observation could determine the multipole moments of a spacetime decreases as more multipoles are included in the model , taken from ryan . results are shown for two typical cases , a 10 m + 10 m inspiral and a 10 m + 10 m inspiral ; in both cases the snr of the inspiral is 10 . ryan s analysis was restricted to circular and equatorial orbits , but a counting argument suggests that spacetime mapping should still be possible for generic orbits . one complication is that the evolution of the orbital elements must also be inferred from the observation , which spoils the nice form of the expansions ( 55)(56 ) , since all the multipole moments now enter at each order of the expansion . however , this would also be true if the expansions for circular - equatorial orbits were rewritten as an expansion in some initial frequency , 0 , which would more closely represent a band - limited observation . in practice , the lowest - order multipole dominates the lowest term in the expansion and so on , which makes spacetime mapping possible in practice . the ryan formalism neatly illustrates why spacetime mapping with emris is possible , but it is not a very practical scheme . we expect that the massive central objects are indeed kerr black holes and so we really want to consider what imprint small deviations from kerr will leave on the emitted gws . a multipole - moment expansion is not a very practical way to do this , as the kerr metric has an infinite number of nonzero multipoles . several authors have adopted the approach of constructing spacetimes given by schwarzschild - kerr plus a small deviation , and have examined the properties of geodesics in those spacetimes . this was constructed by writing the metric in weyl coordinates and adding a quadrupole perturbation to the potential ( in these coordinates , the potential equation reduces to the flat - space laplacian for the ( , z ) cylindrical coordinates , which facilitates writing solutions ) . they considered two types of quadrupole perturbation : a torus around the black hole , and the addition of a point mass at each pole . in the second case , the spacetime necessarily contains line singularities running from the point masses either to infinity or to the black - hole horizon , which are needed to support the point masses . the solutions are perturbative , in that the authors kept only the terms that are linear in the deviation from schwarzschild . collins and hughes explored the properties of orbits in these spacetimes by comparing the precession and orbital frequencies of equatorial orbits in the spacetime to orbits with the same orbital parameters in kerr . they found that there were measurable differences in the perihelion precession in the strong field : for instance , at a radius of 20 m for a 2% perturbation of the black hole , the trajectory would accumulate one radian of dephasing in 1000 orbits . collins and hughes coined the term bumpy black hole to describe spacetimes of this type . starting from the hartle - thorne metric [ 220 , 221 ] , which is an exact solution to einstein s equations describing the spacetime outside of a slowly , rigidly rotating axisymmetric object , the authors constructed a spacetime with metric of the form \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${g_{\mu \nu } } = g_{\mu \nu}^{{\rm{kerr } } } + \varepsilon { h_{\mu \nu } } + o(\delta { m_{l \ge 3}},\,\delta { s_{l \ge 2}})$\end{document } , working perturbatively and keeping only the m2 perturbation in the quadrupole mass moment . a comparison of the frequencies of eccentric equatorial geodesics in the quasi - kerr spacetime to the same geodesics in the kerr spacetime indicated that it would take only 100 cycles to accumulate a /2 phase shift for a 1% deviation from kerr . they also computed waveform overlaps and found that , over the radiation - reaction timescale , the overlap of the waveforms for an orbit with a semilatus rectum of ten geometric radii ( gm / c ) was 20% , 50% , 90% , 98% for inspirals with mass ratio /m = 10 , 10 , 10 , 10 respectively . a third approach to analyzing deviations from the kerr spacetime was considered by gair et al . , who studied geodesic motion in a family of exact spacetimes due to manko and novikov , which include the kerr spacetime for a specific choice of parameters . by using exact solutions of the einstein field equations , they obtained solutions that were valid everywhere , in contrast to the perturbative solutions considered in [ 125 , 207 ] , which break down near the central object . however , this scheme offers less control over the multipole moments , since it is possible to choose the lowest multipole that differs from kerr , but then the higher multipoles must also change . studied observable properties of the orbits , including the variation of the precession frequencies of nearly circular and nearly equatorial orbits as a function of orbital frequency , and the loss of the third integral of the motion ( see section 6.2.5 ) . these three papers outlined different ways to approach the problem of spacetime mapping in practice . however , none of the analyses were complete as they did not consider inspirals . collins and hughes and glampedakis and babak also ignored waveform confusion by assuming that the orbital elements were the same between the orbits under consideration . glampedakis and babak did discuss the importance of confusion and the role of the inspiral evolution in breaking such degeneracies , but no inspiral results were included in the published paper . observationally , the correct orbits to compare will be those with the same frequencies since we have no way to determine the orbital radius or eccentricity directly . assessing the confusion problem is relatively easy , but including inspiral is very hard in general , since the presence of excess multipole moments in the spacetime leads to changes in the rates of energy and angular momentum loss , which must also be included in the analysis . progress can be made in the presence of small deviations by including only the leading - order contributions to the radiation reaction from the multipole moments . this is an open area of research , although barack and cutler carried out a preliminary assessment using post - newtonian emri waveforms augmented with the leading - order contribution of an excess quadrupole moment to the precession and inspiral rates . the resulting waveforms were an improvement in comparison to ryan s analysis , since they included orbital eccentricity and inclination , and were filtered through an approximation of the lisa response . barack and cutler performed a fisher - matrix analysis of parameter - estimation uncertainties , and hence correctly accounted for the confusion issue . for this simple model , they found that a single lisa observation of the inspiral of a 10 m black hole into a 10 m , 10 m or 10 m black hole could measure the deviation from kerr of the quadrupole moment to an accuracy of (m2/m ) 10,10 , 10 , while simultaneously measuring the mass and spin of the central object to fractional accuracies of 10 . this suggests that a lisa - like observatory would be able to perform high - precision tests of the no - hair property of massive compact objects in galactic centers . to put these numbers in perspective , a boson star may have a quadrupole moment that is 100 times that of a kerr black hole with the same mass and spin , so it could easily be excluded . during an emri this can be thought of as a tidal interaction the gravitational field of the small body raises a tide on the horizon that is dragged around through the orbit , leading to dissipation of energy or as energy being lost by gws falling into the black hole . fang and lovelace explored the nature of the tidal - coupling interaction by perturbing a schwarzschild black hole with a distant orbiting moon . they found that the time - dependent piece of the perturbation affected the orbit in an unambiguous way : a time - varying quadrupole moment is induced on the blackhole horizon that is proportional to the time derivative of the moon s tidal field . this quadrupole perturbation extracts energy and angular momentum from the orbit at the same rate that energy and angular momentum enter the horizon . however , the effect of the time - independent piece of the perturbation remained ambiguous . working in the regge - wheeler gauge , fang and lovelace found that this piece vanished , in contrast to a previous result by suen , who used a different gauge . this ambiguity leads to an ambiguity in the phase of the induced quadrupole moment as measured in a local asymptotic rest frame ( larf ) , although the phase of the bulge relative to the orbiting moon is well defined ( using a spacelike connection between the moon and the black hole , fang and lovelace found that the horizon shear led the horizon tidal field by an angle of 4m , where is the angular velocity of the moon ) . the ambiguity of interpretation in the larf makes it impossible to define the polarizability of the horizon or the phase shift of the tidal bulge in a body - independent way . fang and lovelace left open the possibility of developing a body - independent language to describe the response of the central object to tidal coupling , but as yet this has not happened . although the nature of the response of the central object to tidal coupling may be difficult to characterize from gw observations , the total energy lost to tidal interactions is a good observable . ryan s original theorem ignored tidal coupling , but it was later generalized by li and lovelace . they found that the gws propagating to infinity depended only very weakly on the inner boundary conditions ( i.e. , on the nature of the central object ) . this means that the spacetime s multipole structure can be inferred from the outgoing radiation field in the usual way , and hence the expected rate of orbital energy loss , assuming no energy loss into the central body , can be calculated . the rate of inspiral measures the actual rate of orbital energy loss , and the difference then gives the rate at which energy is lost to the central object , which is a direct measure of the tidal coupling . li and lovelace estimated that the ratio of the change in energy radiated to infinity due to the inner boundary condition to the energy in tidal coupling scales with the orbital velocity as v. therefore , it should be possible to simultaneously determine the spacetime structure and the tidal coupling through low - frequency gw observations . information about the central object can also come from the transition to plunge at the end of the inspiral . in a black - hole system , we expect gw emission to cut off sharply as the orbit reaches the innermost stable orbit and then plunges rapidly through the horizon . if there is no horizon in the system , the orbit may instead enter a phase where it passes into and out of the material of the central object . this was explored for boson - star models in : kesden et al . found that persistent radiation after the apparent innermost orbit could be a clear signature of the presence of a supermassive , horizonless central object in the spacetime . this analysis did not treat gravitational radiation or the interaction of the inspiraling body with the central object accurately , but it does illustrate a possible way to identify non - black - hole central objects . something similar might happen if the spacetime were to contain a naked singularity rather than a black hole : in principle , the nature of the emitted waveform after plunge would encode information about the exact nature of the central object . however , this has not yet been investigated . naked - singularity spacetimes may have very - high - redshift surfaces rather than horizons : these spacetimes would be observationally indistinguishable from black holes , unless the inspiraling object happened to move inside the high - redshift surface and then emerged , and the two epochs of radiation could be connected observationally . another example of an object that can be arbitrarily close to a schwarzschild black hole in the exterior but lack a horizon is a gravastar . these are constructed by matching a de sitter spacetime interior onto a schwarzschild exterior through a thin shell of matter , whose radius can be made arbitrarily close to the schwarzschild horizon . it was shown in that the oscillation modes of such a gravastar have quasinormal frequencies that are completely different from those of a schwarzschild black hole . therefore the absence of a horizon would be apparent if ringdown radiation was observed from such a system . in addition , the tidal perturbations that arise during the inspiral of a compact object into a gravastar during an emri can resonantly excite polar oscillations of the gravastar as the orbital frequency passes through certain values over the course of the inspiral . the excitation of these modes generates peaks in the gw - emission spectrum at frequencies that are characteristic of the gravastar , and can also show signatures of the microscopic surface of the gravastar . this process would be apparent both in the amplitude of the detected gws and in the rate of inspiral inferred from the gravitational - waveform phase , since the rate of inspiral will change significantly in the vicinity of each resonance due to the additional energy radiated in the excited quasi - normal modes . although the gravastar model used in [ 346 , 347 ] may not be physically relevant , this work illustrates the more general fact that if the horizon of a black hole is replaced by some kind of membrane , then the modes of that membrane will inevitably be excited during an inspiral and these modes will typically be different to those of a black hole . a change in the inspiral trajectory need not be caused only by differences in the central object , but might arise due to the presence of material in the spacetime , close to the black hole but external to the event horizon . such material could influence the inspiral trajectory , and hence the emitted gws , in two distinct ways : the gravitational field of the matter could modify the multipole moments of the spacetime and hence the orbit as discussed above ; if the orbital path intersected the material , it would cause sufficient hydrodynamic drag on the object to alter the orbit . the influence of the gravitational field of external material was considered in : barausse et al . constructed a model spacetime that included both a black hole and an external torus of material very close to the central black hole . they examined the properties of orbits in two systems : one with a torus of comparable mass and spin to the central black hole ( spacetime a ) , and one with a torus of low mass , but much greater angular momentum than the central black hole ( spacetime b ) . their comparisons were based on computing equatorial geodesics and then kludge gravitational waveforms in the spacetime and in a corresponding kerr spacetime , and then evaluating the waveform overlap . orbits were identified by matching the radial and azimuthal frequencies between the orbits in the two spacetimes in two ways : altering the orbital parameters , while setting the mass and spin to be the same in the corresponding kerr spacetime ; or altering the mass and spin , while keeping the orbital parameters the same . this approach identified a confusion problem : over much of the parameter space the overlaps were very high , particularly when the second approach was adopted . b than in spacetime a , and overlaps for internal orbits between the black hole and the torus were particularly low . this work suggested that it would not be possible to distinguish between such a spacetime and a pure kerr spacetime in low - frequency gw emri observations . the need to constantly adjust the orbital parameters in order to maintain equality of frequencies would lead to a difference in the evolution of the orbit between the two spacetimes , which might break the waveform degeneracies . the torus model was also not physical , being much more compact than one would expect for agn discs . the effect of hydrodynamic drag on an emri was first considered in and was found to be negligibly small for systems likely to be of interest to space - based gravitational - wave detectors . the problem was revisited in : barausse and rezzolla considered a spacetime containing a kerr black hole surrounded by a non - self - gravitating torus with constant specific angular momentum . the hydrodynamic drag consists of a short - range part that arises from accretion , and a long - range part that arises from the gravitational interaction of the body with the density perturbations it causes in the disc . the accretion onto the small object was modeled as bondi - hoyle - lyttleton accretion6 and the long - range force using collisional dynamical - friction results from the literature [ 264 , 50 ] . the effect of the hydrodynamical drag on the orbital evolution was computed for geodesic orbits and compared to the orbital evolution from radiation reaction for a variety of torus models , varying in mass and outer radius . the conclusion was that , for realistic outer radii for the torus , rout > 10 m , the effect of hydrodynamic drag on the orbital radius and eccentricity was always small compared to radiation reaction . the hydrodynamic drag has a greater relative effect on the orbital inclination , and tends to cause orbits to become more prograde , which is opposite to the effect of radiation reaction . for rout = 10 m , the hydrodynamic drag becomes significant at r 35 m , but this radius is smaller for a more compact torus . thus , this effect will only be important for lisa if we observe a system with a very compact accretion torus , or for systems of low central mass . for the latter , the gws are detectable from orbits at larger radii where hydrodynamic drag can be important . however , the snr of such events will be low , so we are unlikely to see many of them . thus , although it seems that this effect is also marginal , this conclusion is based on considering geodesic orbits , and the possible secular build up of a drag signature over the inspiral has yet to be examined , the influence of an accretion disc on the evolution of an emri embedded within it was explored in [ 493 , 268 ] . one of the channels that has been suggested to produce emris is the formation of stars in a disc , followed by the capture of the compact stellar remnants left after the evolution of those stars [ 18 , 16 ] . the migration of such an emri through the accretion disc could potentially leave a measurable imprint on the gw signal . the literature distinguishes between two types of migration . in type i migration , which generally occurs for lower - mass objects the object excites density waves in the disc , which exert a torque on the object . in general , the torque from material exterior to the orbit is greater than that from material interior to the orbit , which causes the object to lose angular momentum and spiral inward on a timescale that is short relative to the lifetime of the disc . in type ii migration , material enters the gap on the disc s accretion timescale , driving the object and gap inwards on that timescale . [ 493 , 268 ] considered migration in geometrically - thin and radiatively - efficient discs , in which thermal energy is radiated on a much shorter timescale than the timescale over which the material moves inward , so the disc can remain thin . such discs can be described by the shakura - sunyaev -disc model , in which the viscous stress in the disc is proportional to the total pressure at each point in the disc . these discs are known to be unstable to linear perturbations [ 286 , 410 , 83 , 358 ] . the alternative -disc model , where stress is proportional to the gas pressure only , is stable to perturbations . yunes , kocsis et al . showed that , over a year of observation , type i migration could lead to 0.01/10 radian dephasings in an emri signal for both -discs and -discs , while type ii migration could lead to dephasings of 10/10 radians . the effects are larger for discs , since these can support higher surface density . for more massive central black holes 10 m the dominant contribution is from bondi - hoyle accretion ( see footnote 6 for a description ) , while for less massive black holes of 10 m the dominant contribution is from the migration . these dephasings were computed for the same system parameters ( apart from maximizing over time and phase shifts ) , so they do not account for possible parameter correlations , but the authors argue that the migration dephasing decouples from gw parameters as the effect becomes weaker with decreasing orbital radius , while relativistic effects become stronger . the relative number of emris that will be produced in discs rather than from other channels is not well understood . however , it will be straightforward to identify such emris , which will be circular and equatorial , to look for and constrain effects of this type . another important question that has not yet been addressed is how to distinguish the effect of external material from a difference in the structure of the central object . if the orbit does not intersect the material , such identification would come from the variation in the effect over the inspiral if the change in the multipole structure comes from material , then at some stage the object would pass inside the matter , and the qualitative effect on the inspiral would be different from that of a central object with an unusual multipole structure . if the orbit does intersect the material , then the spacetime - mapping analysis described above no longer applies , since the geroch - hansen multipole decomposition [ 204 , 217 ] applies to vacuum spacetimes only . if this decomposition could be generalized to nonvacuum axisymmetric spacetimes , then low - frequency gw observation could potentially recover not only the spacetime metric but also the structure of the external material . it would then be possible to verify that this matter obeys the various energy conditions ( see section 2.3 ) . an independent indicator of the presence of material in the spacetime would come from the observation of an electromagnetic counterpart to an emri event . for instance , if an inspiraling black hole was moving through an accretion disc , there might be emission from the material that was accreted onto the inspiraling object or from shocks formed in the disc . again , this has not been explored , although it is likely , given the poor sky - position determination of emri events in gw observations , that it would not be possible to conclusively identify such a weak electromagnetic signature in coincidence with a gw event . the presence of exotic matter outside a black hole , in the form of a cloud of axions , was discussed in . the presence of large numbers of light axions would be one consequence of extra dimensions in string theory , so the detection of an axion cloud would provide strong evidence in support of their existence . the axion cloud would modify the motion of an inspiraling black hole in a similar way to regular matter , although estimates for the precision of measurements possible with future gw detectors have not yet been carried out . the passage of an emri through the cloud could also lead to its disruption , which may rule out the cloud as a possible explanation for any observed deviations , but further theoretical work is required to properly quantify these processes . if multiple states with the same orbital momenta l and magnetic moments m but different principal quantum numbers n are occupied , transitions between these states can generate gws with characteristic strain 59\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\sim{10^{- 22}}{\alpha ^2}{({\epsilon _ 0}{\epsilon _ 1})^{1/2}}\left({{{10\,{\rm{mpc } } } \over r } } \right)\,\,\left({{{{m_{{\rm{bh } } } } } \over { 2\,{m _ \odot } } } } \right)$$\end{document } for a black hole of mass mbh at a distance r. the pre - factor ( 01 ) depends on the axion masses and coupling . the axions can also undergo annihilations , which generate gws with very similar characteristic strains 60\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\sim{10^{- 22}}{\alpha ^7}\epsilon \left({{{10\,{\rm{mpc } } } \over r } } \right)\,\,\left({{{{m_{{\rm{bh } } } } } \over { 2\,{m _ \odot } } } } \right).$$\end{document } in both cases the frequency depends on the black - hole mass in the usual way f 1/mbh , with typical values for 2 m black holes of 100 hz and 30 khz respectively . both elisa and ligo could place interesting constraints on the axion parameter space through ( non)detections of these events . finally , the self - interactions in the axion cloud could eventually lead to the collapse of the cloud in a explosion [ 269 , 489 ] , which would generate gws with strain 61\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$h\sim{10^{- 17}}\left({{\epsilon \over { { { 10}^{- 4 } } } } } \right)\,\,\left({{{100\,{\rm{mpc } } } \over r } } \right)\,\,\left({{{{m_{{\rm{bh } } } } } \over { { { 10}^8}\,{m _ \odot } } } } \right)$$\end{document } and frequency c / gmbh . recent calculations suggest that a bosenova from the milky way black hole at saggitarius a * would be marginally detectable by lisa . the bosenova explosion comes about due to a super - radiant interaction between the cloud of particles and the central black hole , which extracts rotational energy from the black hole and transfers it to the cloud of particles . recent results on these super - radiant instabilities can be found in . in the observable signatures of the presence of a cloud of bosons outside a massive compact object was considered . it was shown that the motion of a particle through the cloud would be dominated by boson accretion rather than by gravitational radiation reaction . during this accretion - dominated phase , the frequency and amplitude of the gravitational - wave emission is nearly constant in the late stages of inspiral . the authors also considered inspirals exterior to the boson cloud , and found that resonances could occur when the orbital frequency matched the characteristic frequency associated with the characteristic mass of the bosonic particles . these resonances could lead to significant , detectable deviations in the phase of the emitted gws . perturbations to the emri trajectory could also arise from the gravitational influence of distant objects , such as other stars or a second mbh . at present , it is thought to be very unlikely that a second star or compact object would be present in the spacetime sufficiently close to the emri to leave a measurable imprint on the trajectory , although detailed calculations for this scenario have not been carried out . however , if the mbh that was the host of the emri was in a binary with a second mbh , this could perturb the trajectory by a detectable amount . the primary observable effect is a doppler shift of the gw signal , which arises due to the acceleration of the center of mass of the emri system relative to the observer . it was estimated that , for typical emri systems , the presence of a second black hole within a few tenths of a parsec would lead to a measurable imprint in the signal . the frequency scaling of the doppler effect differs from the scaling of the post - newtonian terms in the unaccelerated waveform , which suggests that this effect will be distinguishable in gw observations . the magnitude of the leading - order doppler effect scales as msec / r , where msec is the mass of the perturbing black hole , and r is its distance . if the second object is within a few hundredths of a parsec , higher - order time derivatives could also be measured from the gw observations . these scale differently with msec and r , which would in principle allow the mass and distance of the perturber to be measured from the emri data . the probability that a second black hole would be within a tenth of a parsec of a system containing an emri is difficult to assess . at redshifts z < 1 , at any given time a few percent of milky way - like galaxies will be involved in a merger , which suggests an upper limit of a few percent of emris that could have perturbing companions . however , there is uncertainty as to how long the black - hole binary will spend at radii of a few tenths of a parsec following a merger , and it is plausible that the presence of a second black hole would increase the emri rate by perturbing stars onto orbits that pass close to the other black hole , introducing an observational bias in favor of these systems . given these uncertainties , the possibility of a distant perturber will have to be accounted for in the analysis , and , if it is observed in some systems , lisa - like detectors could indirectly inform us of the processes that drive mbh mergers . the gravitational influence of a second stellar mass black hole on the evolution of an emri were considered in . it was shown that a second black hole with a semi - major axis of 10 pc could influence the orbital parameters of an emri ongoing in the same galaxy and already in the low - frequency gw band . it was estimated that in 1% of emris a second black hole could be within the required distance . however , the timescale of the chaotic motion was 10 s , which is much longer than a typical low - frequency observation , and the system considered in had an orbital period of 6 10 s , which would probably not yet be detectable by lisa - like detectors . on the timescale of a gw mission , the effect would probably manifest itself as a linear drift in the orbital parameters , and therefore it is unlikely that this effect would prevent the detection of an emri signal . a full analysis of the effect on parameter estimation has not yet been carried out . it was demonstrated by carter that the kerr metric has a complete set of integrals in addition to the energy and angular momentum that arise as conserved quantities in any stationary and axisymmetric spacetime , geodesics in the kerr metric conserve the carter constant , q. this is the analog of the third isolating integral found in some classical axisymmetric systems , and has been shown to arise due to the existence of a killing tensor of the spacetime . in the schwarzschild limit , q is the sum of the squared angular momentum components in the two equatorial directions . the kerr metric is one of a very special class of metrics that have this property . in fact , carter demonstrated that it was the only axisymmetric metric not containing a gravitomagnetic monopole for which both the hamilton - jacobi and schrdinger equations were separable . the special nature of the kerr metric was emphasized by will who demonstrated that , in newtonian gravity , motion about a body with an arbitrary set of multipole moments ml possesses a third integral of the motion only if the multipole moments obey the conditions 62\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${m_{2l + 1 } } = 0,\quad \,\,{m_{2l } } = m{({m_2}/m)^l}\quad { \rm{for}}\,{\rm{every}}\,l,$$\end{document } which are precisely the conditions satisfied by the mass moments of a kerr black hole , eq . the separability of the kerr metric aids the analysis of inspirals in that spacetime , but it also suggests another potential observable that would show a deviation from the kerr metric in a gw observation . the specialness of the third integral in the kerr spacetime suggests that if a spacetime differed from the kerr solution , even by a small amount , the third integral might vanish , which would potentially lead to the existence of chaotic orbits . if such orbits were observed it would be a clear smoking gun for a deviation from the kerr metric . the standard approach is via construction of a poincar map : a geodesic is computed in cylindrical coordinates ( , z , , ) , and the values of and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\dot \rho}$\end{document } recorded every time the particle intersects a specified plane z = constant . if the resulting plot of all these points on a ( , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\dot \rho}$\end{document } ) plane yields a closed curve , then a third integral exists , otherwise the orbit is chaotic figure 7poincar map for a regular orbit ( left panel ) and a chaotic orbit ( right panel ) in the manko - novikov spacetime . image reproduced by permission from , copyright by aps . poincar map for a regular orbit ( left panel ) and a chaotic orbit ( right panel ) in the manko - novikov spacetime . chaotic motion has been found by sota et al . for orbits in the zipoy - voorhees - weyl and curzon spacetimes ; by letelier and viera for orbits around a schwarzschild black hole perturbed by gws ; by gueron and letelier for orbits in a black - hole spacetime with a dipolar halo , and in prolate erez - rosen bumpy spacetimes ; and by dubeibe et al . for some oblate spacetimes that are deformed generalizations of the tomimatsu - sato spacetime the only investigation to date of chaotic orbits in the context of lisa was by gair et al . , who explored geodesic motion in a family of spacetimes due to manko and novikov that had arbitrary multipole moments , but which included kerr as a special case . considered orbits in a family of spacetimes parameterized by a single excess quadrupole moment they found that , while the majority of orbits in these spacetimes possessed an apparent third integral , chaotic orbits existed very close to the central object for arbitrarily small oblate deformations of the kerr solution . as the spacetime was deformed away from kerr , a second allowed region for bound geodesic motion was found to appear close to the central black hole , in addition to the allowed region present in the kerr metric . concluded that this chaotic region would probably be inaccessible to an object that was initially captured at a large distance from the central object . this analysis was revisited in but the conclusions in that paper were the same . the only difference was that the authors in identified a region of stable motion within the inner region that contains chaotic orbits . the chaotic orbit shown in the right hand panel of figure 7 appears to pass in and out of the region of stability which should not happen , so this might be a numerical artifact . however , the existence of chaotic orbits and the probable inaccessibility of these orbits to inspirals was confirmed by . brink also explored integrability in arbitrary stationary , axisymmetric and vacuum spacetimes , concentrating on regions where the poincar maps indicated the presence of an effective third integral . brink hypothesized that some spacetimes might admit an integral of the motion that was quartic in the momentum , in contrast to the carter constant , which is quadratic . the kolmogorov , arnold , and moser ( kam ) theorem indicates that when a hamiltonian system with a complete set of integrals is weakly perturbed , the phase - space motion will either be confined to the neighborhoods of the invariant tori , or the motion will be chaotic . thus , if there is a region of the spacetime where chaotic motion exists , there can not be another region where a third invariant exists . a mathematical demonstration that the orbits can possess an approximate invariant , while technically being chaotic , is lacking at present , small everywhere , since the change in mutipole moments necessarily changes the horizon structure and so the perturbation is infinitely large at certain points . a discussion of the reason for the existence of chaotic geodesics in some spacetimes was given by sota et al . . they suggested that it would arise either from a change in sign of the eigenvalues of the weyl tensor at a point , which would lead to a local instability , or from the existence of homoclinic orbits . the latter explanation applied only to non - reflection - symmetric spacetimes , while most spacetimes of astrophysical interest should be reflection symmetric . the weyl - tensor analysis has not been carried out for the manko - novikov family of spacetimes . one other proposed explanation was the existence of a region of closed timelike curves , which was found to touch the region in which chaotic motion was identified . in conclusion , it seems unlikely that chaotic geodesics will be found in nature , but if they were identified we would know immediately that the spacetime was not kerr . one possibility would be to observe the transition from regular to chaotic motion in a time - frequency analysis : the regular motion would be characterized by a few well - distinguished peaks in a fourier transform of the signal , while chaotic motion would show a much broader band structure . however , it is not clear that it would be possible to distinguish the chaotic phase from detector noise , and hence there would be no way to identify an inspiral that ends by entering a chaotic phase as opposed to one which ends at plunge into a black hole . if an orbit passed into a chaotic phase and then back into a regular phase we might see a signal turn on and off repeatedly . however , the chaotic motion would randomize the phase at the start of the regular motion , so to detect such a signal we would need each regular phase to be long enough that they were individually resolvable by matched filtering . eccentric and inclined emris will generically pass through transient resonances at which the radial and frequencies become commensurate . for emris in gr , these resonances will be isolated , and the transition through resonance will proceed on the usual radiation - reaction timescale but with a temporary modification in the energy flux on resonance . however , according to the poincar - birkhoff theorem , when an integrable system is perturbed it causes the appearance of a birkhoff chain of islands whenever the frequencies of the system are at resonance . therefore , in a perturbed kerr spacetime , another observable consequence would be that the emri frequencies could remain on resonance for many more cycles , providing another smoking gun for a deviation from kerr [ 24 , 294 ] . detection of a persistent resonance in a matched - filtering search will require a modification of the search pipeline , but it should be considerably more straightforward than detection of chaos , as the signal will be coherent and could therefore be identified using time - frequency methods , or a phenomenological waveform model . as mentioned in section 5.1.3 , in massive scalar - tensor theories , a different type of persistent resonance can occur , in which a super - radiant scalar flux balances the gw flux [ 110 , 495 ] . such resonances can last a significant fraction of a hubble time and so observing a single emri offers significant constraining power on the space of massive scalar - tensor theories . this is not a test of black - hole structure , since the resonance is between the scalar and gravitational fluxes , rather than in the geodesics of the central object , but the observable effects are similar so we mention it here . the ryan mapping approach uses observations of precession frequencies as functions of the orbital frequency to extract the spacetime metric from gws . however , even if the metric is found to be kerr , this is not enough to verify gr . it was pointed out by psaltis et al . that the kerr metric is a solution to the field equations for several alternative theories of gravity . essentially , since the kerr metric has vanishing ricci tensor , r = 0 , any theory in which the vacuum field equations depend only on r will also admit kerr as a solution . allowing for a nonzero cosmological constant , a , a black - hole solution in gr satisfies 63\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${r_{\mu \nu } } = { r \over 4}{g_{\mu \nu}},\quad r = 4\lambda , \quad { \rm{and}}\quad { r_{;\mu } } = 0,$$\end{document } in which r denotes the ricci scalar and is the spacetime metric . discussed four different alternative theories , already described in section 2.1 f(r ) gravity in the metric formalism . if f(r ) is expanded as a taylor series a0 + r + a2r + , there are three possible cases . ( i ) if a0 = 0 the kerr solution , which corresponds to r = 0 , is always a solution to the equations of motion . ( ii ) if the taylor series terminates at a2 , all constant curvature solutions of gr ( with any ) remain exact solutions of the f(r ) theory . ( iii ) if a0 0 and the series does not terminate at a2 , constant - curvature solutions of gr will be solutions of the f(r ) theory with different values of the curvature . the difference in curvature will be small , however.f(r ) gravity in the palatini formalism . in this case , any constant - curvature solution of gr is also a solution to these equations , with the same christoffel symbols . this is unsurprising , as it is known that palatini f(r ) gravity reduces to gr in vacuum .general quadratic gravity . for any black - hole solution in gr , the tensors and that appear in the field equations ( 18 ) both vanish , and the field equations reduce to those of gr . hence all black - hole solutions of gr are solutions in this theory.vector-tensor gravity . in this case , we find once again that all constant - curvature solutions of gr are solutions to the equations , but with a shifted value of the curvature that depends on the vector field strength r = 16/(4 + ( 4 + 3)k ) . if f(r ) is expanded as a taylor series a0 + r + a2r + , there are three possible cases . ( i ) if a0 = 0 the kerr solution , which corresponds to r = 0 , is always a solution to the equations of motion . ( ii ) if the taylor series terminates at a2 , all constant curvature solutions of gr ( with any ) remain exact solutions of the f(r ) theory . ( iii ) if a0 0 and the series does not terminate at a2 , constant - curvature solutions of gr will be solutions of the f(r ) theory with different values of the curvature . any constant - curvature solution of gr is also a solution to these equations , with the same christoffel symbols . this is unsurprising , as it is known that palatini f(r ) gravity reduces to gr in vacuum . general quadratic gravity . for any black - hole solution in gr , the tensors and that appear in the field equations ( 18 ) both vanish , and the field equations reduce to those of gr . hence all black - hole solutions of gr are solutions in this theory . vector - tensor gravity . in this case , we find once again that all constant - curvature solutions of gr are solutions to the equations , but with a shifted value of the curvature that depends on the vector field strength r = 16/(4 + ( 4 + 3)k ) . the action for general quadratic gravity introduced by stein and yunes also admits the kerr solution , but only in the non - dynamical version of the theory in which the functions are constants . in that case the field equations are once again satisfied by spacetimes with rab = 0 , and so the vacuum solutions of gr are solutions to the field equations in these theories as well . in the dynamical version of the theory , the riemann tensor enters the field equations explicitly and so rab = 0 is no longer sufficient to satisfy them . although it is true that all of these theories admit the kerr metric as a solution , this does not mean that we have no way to distinguish between them via gw observations . this was not discussed in , but is argued in a comment on that paper by barausse and sotitirou . first , the uniqueness theorems of gr [ 351 , 224 , 248 , 114 , 379 ] do not necessarily apply in these alternative theories . in other words , just because the kerr metric is a solution does not mean that we would expect it to form as a result of gravitational collapse . this is an equally important consideration as to what we would expect to see in the universe , although this argument can be sidestepped by suitable fine tuning . in f(r ) gravity , the metric of a spherically - symmetric body is not the schwarzschild metric , but has a yukawa correction . the constraints that lisa could place on such a deviation from the kerr solution were investigated in . expanding f(r ) to quadratic order ( r + a2r/2 ) , it was found that emri observations could place a bound |a2| 10 m , about an order of magnitude better than the bound from observations of planetary precession in the solar system , |a2| < 1.2 10 m. however , the bound from the et - wash laboratory experiments is many orders of magnitudes better , |a2| < 2 10 m [ 116 , 66 ] . the gw constraints will be obtained in a very different curvature regime and could be of interest if something like the chameleon mechanism is invoked . the chameleon mechanism was introduced to allow f(r ) models to explain cosmological acceleration without violating laboratory and solar - system constraints . it is a nonlinear effect that could arise when the curvature is very different from the background value , e.g. , in the vicinity of matter . if the matter density is high the scalar degree of freedom in f(r ) gravity acquires an effective mass , which means that the effective coupling to matter becomes much smaller than the bare coupling , which is relevant on cosmological scales . therefore the bare coupling could be much higher than inferred from laboratory constraints , allowing the theories to explain cosmological acceleration ( see for a full description of the mechanism and complete references ) . in a similar way , the effective coupling in the vicinity of a compact object could in principle be different from that in the laboratory and so the weak constraints from gravitational - wave observations are still interesting because they probe a different curvature scale . subsequent to the publication of , it has been shown that the end state of gravitational collapse in f(r ) theories ( and scalar - tensor theories ) is not the point - mass limit of an extended body , but is in fact the kerr solution . therefore the results in do not apply to black holes in f(r ) gravity , but would apply for an exotic horizonless compact object if one existed . the constraints that low - frequency gw could place on this metric can also be considered to be constraints on a strawman yukawa - like deviation from the kerr solution , without reference to a specific theory in which such corrections arise . gws are generated by perturbations of the spacetime and hence depend on the full dynamical sector of the theory . therefore the response to perturbations will be different if the field equations are different , even if the unperturbed metric is the same . in it was demonstrated that , for metric f(r ) gravity , linearizing about the minkowski spacetime gives rise to massive graviton modes in addition to the standard transverse - traceless modes of gr . these can not be zeroed out by a gauge transformation . to date , no one has considered the generation of such massive modes in a binary system nor the observational consequences . however , if these modes are generated , there would be two natural ways to find them : a direct gw detection ( the modes have different polarization states and propagation velocities ) , and an indirect detection , as the binary system would inspiral faster than expected due to loss of additional energy in the extra modes . it is not clear whether the latter will be distinguishable from tidal interaction effects , and quantitative estimates of the power of tests that would be possible with lisa - like detectors have not yet been done . a second example of an alternative theory in which a spacetime metric is the same as a gr solution but behaves differently perturbatively is dynamical chern - simons ( cs ) gravity . the nonrotating - black - hole solution in that theory is schwarzschild , as in gr , but it was shown in that there are 7pn and 6pn corrections respectively to the scalar and gravitational energy flux radiated to infinity by a circular emri . this could lead to a 1-cycle difference in waveform phasing over a year of inspiral if the cs coupling parameter || 0.1 . in post - newtonian results were obtained for the emission from spinning black - hole binaries in a general quadratic - gravity theory . one model within the general class considered corresponds to dynamical cs gravity and for that case the results were found to be consistent with the perturbative calculations in . in interpreting gw observations , it will be necessary to verify both the static and dynamic aspects of the theory . the ryan mapping algorithm is the natural way to start , but if a metric is found to be consistent with kerr it will then be necessary to verify that the observed gws and energy loss are also in agreement before concluding that we are observing a system consistent with a kerr black hole in gr . certain alternative theories of gravity do not admit the kerr metric as a solution : these include the dynamical version of general quadratic gravity described by eq . it is difficult to solve the field equations in alternative theories , so few analytic black - hole solutions are known outside of gr . solutions for slow rotation are known in dynamical cs gravity , which is a special case of eq . ( 19 ) with 1 = 2 = 3 = 0 , to leading order in spin and now to quadratic order . slow - rotation solutions are also known in einstein - dilaton - gauss - bonnet ( edgb ) gravity , in which gravity is coupled to dilaton and axion fields . in the weak - coupling limit , spherically symmetric and stationary solutions to the general quadratic - gravity theory described by eq . ( 19 ) take the same form as the solutions to edgb gravity and were derived in . solutions to this same class of theories are also known for arbitrary coupling , but only under the slow rotation approximation , and these were given in . in dynamical cs gravity , it was shown that the metric describing a slowly rotating black hole at linear order in the spin differs from the slow - rotation limit of the kerr metric beginning at the fourth multipole , l = 4 . it was also shown that the equations describing perturbations of this metric in dynamical cs gravity coincide with those of gr at leading order in spin and coupling parameter , with corrections due to the sourcing of metric perturbations by the scalar field entering at higher order . therefore it was argued that the deviations from gr will manifest themselves primarily through differences in the geodesic orbits in the spacetime . therefore this is completely analogous to spacetime mapping within gr , as described in section 6.2.1 , so the prospects for detection of such deviations with lisa - like detectors are comparable . the energy - momentum tensor of the gws was also shown to take the same form in terms of the metric perturbation as it does in gr , so the leading - order correction to the gw energy flux is determined by these modifications to the conservative dynamics . energy balance also applies in this context and can be used to relate the adiabatic evolution of an orbit to the flux of energy and angular momentum at infinity . however , energy and angular momentum are also carried to infinity by the scalar field , and these corrections to the evolution were not estimated in . considering non - inspiraling geodesic orbits , sopuerta and yunes estimated that imri or emri events observed by lisa would be able to put constraints on the coupling parameter of dynamical cs gravity of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\xi ^{1 \over 4 } } \lesssim { 10 ^ 2}$\end{document } km . a more complete study that included inspiral and used a fisher matrix analysis to account for parameter correlations is described in [ 103 , 104 ] . there it was found that lisa observations of emris could place a bound \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\xi ^{{1 \over 4 } } } < { 10 ^ 4}$\end{document } km . this is somewhat weaker than the bound estimated for imris in , but better than the estimate for emris in that paper , so imri systems ( involving a 10 m object inspiraling into a 10 m object ) may be able to place even more stringent constraints than originally estimated , if they were observed . the emri bound \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\xi ^{{1 \over 4 } } } < { 10 ^ 4}$\end{document } km is four orders of magnitude better than the best solar - system bound , which is based on data from gravity probe b and lageos satellites [ 169 , 122 ] . in the bound from current binary - pulsar observations was estimated to be four orders of magnitude better than that in the solar system and hence comparable to the expected gw result . however , this bound was based on an upper limit on the rate of precession . it was argued in that an upper bound could not place a constraint on the cs deviation since the sign of the cs contribution is opposite to that in gr , and so a bound could only be placed if a lower bound on the precession lying below the gr value could be found . therefore the solar - system bound is the best current constraint , which gw observations will improve by several orders of magnitude . the chern - simons black - hole metric has recently been derived to quadratic order in the spin , so the results discussed here can now be extended to this higher - order metric . this second - order solution had several interesting properties , in particular the petrov type7 is type i , whereas it was type d in the linear - spin case ; furthermore , there is no second - order killing tensor , which means orbits do not have a third carter - constant - like integral of the motion . this could have important observable consequences for gws from emris , as discussed in section 6.2.5 . the analogous calculation to , the evolution of an emri in the strong - field region of the metric , has not yet been carried out for the second - order cs metric . however , the gw emission from quasi - circular binaries in this theory has been determined without any restriction on the spin magnitude or coupling , but in the post - newtonian limit , i.e. , with a restriction on the magnitude of the velocity . this waveform model was constructed using energy balance and expressions for the gravitational - wave energy flux emitted in general quadratic - gravity theories that were derived in . these were given in section 5.1.3 and are two orders of magnitude better than the emri constraints estimated using the linear - spin metric . these constraints were derived in a very different mass - ratio regime ( a binary with mass ratio of 1:2 ) , but the fact that they are so much better is probably not surprising since the quadratic in spin corrections to the metric enter at a lower post - newtonian order than the linear - spin corrections . therefore it is likely that the bounds that emris could place on cs gravity are much stronger than quoted here . this possibility and possible qualitative signatures of the loss of the third integral in the higher - order cs metrics should be further investigated . in the small - coupling limit , it can be shown that the energy - momentum tensor of gws in the general quadratic - gravity theory [ eq . ( 19 ) ] also follows the same quadrupole formula as in gr . using this result , a post - newtonian expression for the energy flux emitted from a quasi - circular binary in these theories corrections to the energy flux come from changes to the conservative dynamics , i.e. , to the orbits that test particles follow in the background metric , from energy lost in scalar field radiation and from the contribution to the gw energy - momentum integral from metric perturbations sourced by the scalar field . the authors found that , for these theories , the scalar dipole radiation dominates the correction to the energy flux and enters at a post - newtonian order of i.e. , at a power of v / c one order before the gw energy flux of gr . there are also 0pn and 2pn corrections to the flux from the scalar - metric interaction and the modification to the conservative dynamics . the observability of these waveform differences with space - based gravitational detectors has , so far , only been directly estimated for the specific case of einstein - dilaton - gauss - bonnet gravity . in that case , the constraints derivable with space - based gw detectors were compared to those that can be obtained from observations of low - mass x - ray binaries . some of these are observed to have orbital decay rates that are larger than predicted by radiation reaction in gr . assuming this excess orbital - decay rate arises from additional scalar radiation emitted from the binary , it is possible to obtain a bound six times stronger than that derivable from solar - system experiments ( using the binary a0620 - 00 ) of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\sqrt { |\alpha | } < 1.9 \times { 10 ^ 5}$\end{document } cm . elisa would be able to place a bound that is slightly stronger ( a factor of two ) than this , while combining multiple decigo observations would yield a bound three orders of magnitude better . for other theories in this general quadratic class , however , the post - einsteinian parameters have been calculated for circular - equatorial inspirals in this class of theories . the parameters characterizing the modification to the gr waveform in the ppe framework are defined through the expression 64\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\tilde h = { \vert}{\tilde h_{{\rm{gr}}}}{\vert}(1 + \alpha { \eta ^c}{u^a})\exp [ { \rm{i}}\psi _ { { \rm{gw}}}^{{\rm{gr}}}(1 + \beta { \eta ^d}{u^d})],$$\end{document } in which = m1m2/(m1 + m2 ) is the mass ratio of a binary with component masses m1 and m2 , u = f , = (m1 + m2 ) is the chirp mass and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\tilde h}_{{\rm{gw}}}}/\psi _ { { \rm{gw}}}^{{\rm{gr}}}$\end{document } are the general - relativistic waveform and waveform phase respectively . under the weak - coupling approximation , the metric of non - spinning black holes in these general quadratic - gravity theories depends only on a parameter \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$\varsigma \propto \alpha _ 3 ^ 2$\end{document}. for mergers of such black holes , the ppe parameters were found to be = ( 5/6) , = ( 50/3) , a = 4/3 = b , c = 4/5 = d . more recently , the ppe corrections to the waveform phase ( which are parametrized by b and ) were found without making a weak - coupling approximation and for binaries with spinning components . the exponent depends on whether the black holes are spinning and on which coefficients of the theory are allowed to be non - zero . the authors considered the cases of odd parity ( i.e. , including the pontryagin term 4 0 ) and even parity ( i.e. , including the other terms quadratic in the curvature , 1 , 2 , 3 = 0 ) separately . the exponent of the ppe phase correction was found to be b = 7/3 for spinning black holes in even - parity theories , b = 1/3 for spinning black holes in odd - parity theories , and b = 3 for non - spinning black holes . the authors also obtained equations , which we do not present here , relating the ppe amplitude , , to the coupling constants of the theory . if a gw observation is used to place bounds on these ppe parameters , these expressions can be used to translate the bound into a constraint on this set of alternative theories of gravity . as discussed in section 5.2.2 , the authors of considered more general possible forms for black - hole solutions without specifying a particular alternative theory . by imposing the existence of a carter - like third integral of the motion , asymptotic flatness , and the peeling theorem8 , and by setting as many metric components to zero as possible while still recovering all known modified gravity spacetime metrics , they obtained a family of generic modified black - hole solutions . it was subsequently realized that all solutions admitting a carter constant had been found in , but identifies the physically - realistic subset of these general solutions . this approach is suited to the construction of eccentric , inclined inspirals , and in the gws for generic emris occurring in these metrics were constructed by extending the analytic - kludge framework . as yet , these waveforms have not been used to assess the ability of a space - based detector to constrain such deviations from the kerr solution , but work in this area is ongoing . the previous sections 6.2.16.2.6 have identified some of the possible causes of deviations in the structure of the central object from the kerr metric , as well as some observational consequences of these deviations . nevertheless , interpreting gw observations correctly is a nontrivial challenge . our working assumption is that the massive compact objects that occupy the centers of most galaxies are indeed kerr black holes . therefore it is reasonable to design an approach to spacetime mapping that looks for inspirals into kerr black holes and quantifies any deviations from such inspirals that may be present . the starting point is to assume that gr is correct , and that the source s spacetime is vacuum and axisymmetric . the multipole moments can be extracted from the precession frequencies via ryan s algorithm , and then the expected gw and inspiral rate for such a spacetime in gr can be computed . if the observations are consistent there is no evidence to contradict the initial assumptions , but we can still do several tests , by asking the following questions : ( i ) does the no - hair if there is a deviation , the spacetime must either contain closed - timelike curves , or lack a horizon . ( ii ) does the emission cut off at plunge or does the radiation persist ? ( iii ) is the tidal interaction consistent with a kerr black hole ? if the radiation still cuts off at a plunge and the multipole structure is not consistent with the kerr metric , it might indicate the presence of a naked singularity , which would be a violation of the cosmic - censorship hypothesis . if the observations are not consistent with gr , the first assumption to relax would be that the spacetime was vacuum . in principle , it might then be possible to deduce both the multipole moments of the spacetime and the energy - momentum tensor of the matter distribution from the observations . the gw could then be computed for such a spacetime in gr , and checked for consistency with the observations . if the observations are consistent , similar questions can be asked : ( i ) does the emission cut off at plunge or does the radiation persist ? ( iii ) does the matter distribution obey the strong , null , and weak energy conditions , or have we identified an exotic matter distribution ? only if the observations are inconsistent will there be evidence of a failure in gr itself . the main complication to this approach is that the detection of emris will rely on matched filtering using waveform templates . if a system differs significantly from a kerr inspiral , then it may not be picked out of the data stream . she suggested that the spacetime - mapping problem could be thought of as analogous to inversescattering problems in quantum mechanics , where the potential to be determined is the ernst potential ( see discussion in section 6.2.1 ) , but the technical details of such a method have not yet been worked out . brink also suggested an approach with a similar philosophy to what we discuss above : assuming that the instantaneous geodesic is triperiodic ( i.e. , that it has a complete set of integrals ) , there is a good chance it will have a high overlap with a kerr geodesic . the inspiral defines a point in the kerr geodesic space , and so matched filtering with kerr snapshots can pick out an inspiral trajectory . the inspiral trajectory in the kerr spacetime can be computed , and we can check to see if the observed inspiral deviates from the predicted one . this approach has two drawbacks : first , it relies on the gws from a geodesic in an arbitrary spacetime having a high overlap with those from at least one kerr geodesic ; second , the interpretation of deviations in the trajectory relies on being able to compute gw emission in alternative metrics in gr , or in alternative theories of gravity . one approach to addressing the first of these problems is to search for emris using a template - free approach . one method would be to search for tracks of excess power in the time - frequency domain [ 194 , 200 , 467 ] . however , excess - power algorithms lose information on the phase of the waveform , which is the most important observable for carrying out tests of gr . more recently , a method that attempts to match the phase of individual harmonics using a taylor expansion with a small number of coefficients was described in , which built on ideas used to search for gr emris in the mock lisa data challenges . while the method appears promising , it must be kept in mind that so far these approaches have only been used on highly simplified datasets containing a single emri source . issues related to confusion between the harmonics of the multiple sources expected in real data have not yet been properly explored . , it is clear that low - frequency gw detectors will be able to check that an emri is consistent with kerr , which can be interpreted as a statement on how much the spacetime could differ from the kerr metric [ 18 , 47 ] and still be consistent with our search templates . what is more difficult is to say exactly what lisa will tell us if we observe something that does differ from the kerr metric . more work is still needed in this area . another class of low - frequency gw sources that are closely connected to emris are extreme - mass - ratio bursts emrbs . these are the precursors to emris : in the standard emri formation channel , compact objects are perturbed onto orbits that pass very close to the central black hole , emitting bursts of gws as they do , which can lead to capture if sufficient orbital energy and angular momentum are lost to gws . furthermore , just after capture , emri compact objects proceed on very eccentric orbits that radiate in bursts near pericenter rather than continuously . emris objects that only encounter the central black hole a few times before being perturbed onto an unbound or plunging orbit . for a sufficiently nearby system ( in the virgo cluster or closer ) , these bursts could be individually detected by lisa - like detectors . the low snr of such events compared to emris is partially outweighed by the significantly larger number of compact objects undergoing flybys at a given time , so the detection rate could be as high as 18 yr , of which 15 yr would originate in the milky way . a more careful analysis predicted only 1 event per year , and a proper analysis of our ability to identify such bursts in the presence of signal confusion has not been carried out . therefore it is unclear at present whether any of these events would be identified in the data of lisa - like detectors . it has been suggested that emrbs could be used to test black - hole structure in a similar way to emris . this assertion was based on comparing approximate burst waveforms coming from spinning and nonspinning central black holes with the same system parameters . in , an analysis of parameter estimation for emrbs accounting for parameter correlations was performed , which concluded that a space - based detector like lisa could measure many of the parameters of the system to moderate precision , including the mass and spin , provided that the periapse of the burst orbit was within 10 gm / c . this analysis did not consider sky position or eccentricity to be free parameters , taking the former to be the center of the milky way , and the latter to be maximal , and it treated the distance and compact - object mass as a single amplitude parameter m / d . the small object mass can not be separately determined because there is no significant orbital evolution over the duration of one burst . the analysis did not consider constraints on gr through measurements of other multipoles , but it was found that the mass and spin could both be measured to high accuracy for orbits that pass sufficiently close to the black hole . therefore it may also be possible to place some kind of constraint on gr deviations using these systems . however , the event rate for such relativistic systems is very low so we will have to be lucky to see a useful event . useful burst events might also be seen from nearby galaxies other than our own , which are thought to have black holes in the appropriate mass range . other unknown black holes in the local universe could also provide sources , but for such extra - galactic systems , the sky position will be unknown and so a single burst will not provide enough information to allow the determination of all the system parameters . if an emrb was observed to repeat several times over the lifetime of a gw mission , then more information could be derived about the system . however , there are serious practical issues with associating multiple , well - separated bursts with the same source . the inspiral orbit might also be perturbed between pericenter passages by encounters with other stars . the prospects for doing useful tests of black - hole structure with emrbs therefore seem rather bleak . moreover , any tests that can be carried out will inevitably be much weaker than those based on emris , as an emrb event will generate several waveform cycles rather than the several hundred thousand from an emri . when a black hole is perturbed , it rapidly settles back down to a stationary kerr state via the radiation of exponentially damped sinusoidal gws known as quasi - normal modes ( qnms ) . for a kerr black hole in gw , the qnm frequencies and damping times are uniquely determined by the mass and spin of the black hole . observations of two or more qnms from the same system will therefore test that the end - state of the merger is a kerr black hole , if they provide consistent estimates for the mass and spin of the central object . qnms will be excited during the merger of supermassive black holes and will dominate the late - stage radiation , so lisa - like detectors should observe qnm radiation with relatively high snrs [ 182 , 155 , 77 ] . in fact , the snr from the qnms alone may be comparable to the total snr in the inspiral . this is illustrated in figure 8 , which shows the snr with which lisa would detect the inspiral and ringdown for equal - mass , nonspinning mbh binaries as a function of black - hole mass . the ringdown dominates the snr for redshifted masses greater than 3 10 m. figure 8comparative snrs , as a function of redshifted black - hole mass ( 1 + z)m , for the last year of inspiral of an equal - mass mbh binary and for the ringdown after the merger of the system . the method used to generate this figure follows that of , updated to use a modern lisa sensitivity curve with a low - frequency cutoff of f = 1 10 hz . the redshift is set to z = 1 , at which the luminosity distance is dl = 6.6 gpc using wmap 7-year parameters . comparative snrs , as a function of redshifted black - hole mass ( 1 + z)m , for the last year of inspiral of an equal - mass mbh binary and for the ringdown after the merger of the system . the method used to generate this figure follows that of , updated to use a modern lisa sensitivity curve with a low - frequency cutoff of f = 1 10 hz . the redshift is set to z = 1 , at which the luminosity distance is dl = 6.6 gpc using wmap 7-year parameters . reviews of the theory of quasi - normal modes can be found in kokkotas and schmidt , in nollert and in berti , cardoso and starinets , but we will briefly summarize the relevant results here . at late times , the gravitational radiation from a perturbed black hole can be written as a sum of exponentially - damped sinusoids . it is known that qnms do not form a complete set in a mathematical sense , but numerical results confirm that the late - time behavior is well described by such an expansion . the angular dependence can be decomposed into a sum of spheroidal harmonics of spin weight 2 , which are labeled in analogy to standard spherical harmonics . for each ( l , m ) , there are infinitely many resonant qnms , which can be labeled in order of decreasing damping time by a third parameter n. the waveform expansion takes the form 65\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${h _ + } + { \rm{i}}{h _ \times } = { m \over r}\sum\limits_{lmn } { { { \mathcal a}_{lmn}}{{\rm{e}}^{{\rm{i}}({\omega _ { lmn}}t + { \phi _ { lmn}})}}{{\rm{e}}^{- t/{\tau _ { lmn}}}}{s_{lmn } } } , $ $ \end{document } where lmn , lmn , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal a}_{lmn}}$\end{document } , and lmn are the frequency , damping time , amplitude , and initial phase of the mode , and slmn is a complex number obtained by evaluating the spheroidal harmonics for the particular orientation and mode frequency . the frequency and damping time , lmn and lmn , are determined solely by the mass and frequency of the central black hole and are the key observables . the damping time can also be considered as the reciprocal of the imaginary part of a complex mode frequency , the amplitude and phase depend on the exact nature of the perturbation that excited the qnms , so they are less useful . one complication is that the mode numbers that are excited [ i.e. , the ( l , m , n ) parameters ] are not known a priori . in the first analysis of snr from qnms , it was assumed that only the l = m = 2 , n = 0 was excited , which simplifies analysis of the observations . however , this simplifying assumption is not needed in general . the use of qnms as a probe of black - hole structure was first proposed by dreyer et al . , who coined the phrase black - hole spectroscopy for this technique . a measurement of lmn and lmn for a single qnm will give several discrete solutions for the black - hole mass and spin , corresponding to different choices for the mode numbers . a measurement of a second qnm will give another discrete set of values , and in general these will not be the same as the first set except for one combination , which are the true values . this not only determines the black - hole parameters , but provides a test that the central object is a kerr black hole , since the relationship between black - hole parameters and frequencies depends on that assumption . if the object was something else , we would find no consistent combination of mass and spin parameters to arise from the analysis . we denote a set of choices for the n modes to which the frequencies correspond by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal q}$\end{document}. each \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal q}$\end{document } defines a two - dimensional curve in the 2n + 2 dimensional space , parameterized by ( a , m ) . the probability distribution for the observed frequencies , , may be denoted by \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$p(\omega |a,\,m,\,{\mathcal q})$\end{document } and can be used to define a confidence interval by determining the p0 such that 66\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$\int\nolimits_{\{\omega : p(\omega { \vert}a , m,{\mathcal q } ) > { p_0}\ } } \,\,p(\omega { \vert}a , m,{\mathcal q}){\rm{d}}{\omega ^{2n}}.$$\end{document } dreyer et al . deemed that an observation was inconsistent with gr if the actual observed frequencies lay outside such confidence intervals for all possible choices of a , m and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${\mathcal q}$\end{document}. using a toy model for a non - black - hole source , and assuming that two qnms were observed , drawn from four possible excited modes , they estimated that , with a false alarm probability of 1% , the false dismissal rate would be 60% if the snr in the weakest mode was 10 , falling to 10% at an snr of 100 . assuming a reasonable amount of energy deposition into the qnms , they estimated that this would be satisfied by all lisa mbh merger sources , so the prospects for such tests are good . they computed snrs that improved on those in by using an up - to - date detector noise curve and including black - hole spin . for a source of mass 10 m < m < 10 m at a distance of 3 gpc , they found snrs between 10 and 2 10 for an excitation energy of 3% of the rest mass , and between 1 and 3000 for an excitation energy of 0.1% . there is little dependence of the snr on the central black - hole spin . putting the source at higher z shifts the sensitive range of masses in the usual way , but even at redshift z = 10 the maximum snr was found to be several hundred . . also looked at parameter - estimation accuracy from observations of one qnm ( with known mode numbers ) and found that lisa observations would measure the mass and spin to fractional accuracies in the range 10 10 for sources at d = 3 gpc and for the masses as quoted above . also performed a fisher - matrix analysis of a two - mode problem , which accounted for all correlations between the modes . pseudo - orthonormal modes with different angular dependence ( l l or m m ) and overtones that is , modes with the same angular dependence ( l = l and m = m ) . in both cases , the accuracy of determination of the mass and spin was comparable to the results quoted for a single mode , although the parameter space was simplified significantly to just five parameters : a , m , two mode amplitudes \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}${{\mathcal a}_1},\,{{\mathcal a}_2}$\end{document } and a common initial phase . berti et al . define two distinct qnms to have resolvable frequencies if the difference in these is larger than the error in both ( and similarly for the damping times ) . by recasting the two - mode model in terms of the two mode frequencies and damping times rather than a and m considered two cases ( the resolvability of either frequencies or damping times , and the resolvability of both ) , and they computed the source snr needed in each case . when the two modes are overtones , the snr required to resolve either was 100 , the exact value depending on the black - hole spin and the ( l , m ) numbers of the mode , but the snr required to resolve both was 1000 . when the two modes are pseudo - orthonormal , the snr range to resolve either is a few if the modes differ in l , and a few tens if the modes have the same l but differ in m and the central black - hole spin is a > 0.4 . there is a mode degeneracy , which means the snr required blows up when l = l and a 0 . the snrs required to resolve both modes are in the range 10010 000 . conclude that even under pessimistic assumptions about the amount of energy radiated in ringdown radiation , it should be possible to resolve one qnm and either the damping time or frequency of a second qnm , provided that the first overtone radiates 10 of the total ringdown energy . this will provide enough for a test of the no - hair property of the central object . a stronger test would come from detecting frequencies and damping times for both qnms , but this would require ringdown snrs 1000 , which is rather unlikely . the main uncertainty in this analysis was in the excitation coefficients for the various modes , but numerical relativity simulations have now provided some information on the excitation of ringdown modes in a merger . this paper revised downward the snr required to detect either the frequency or damping time of a second mode , which is sufficient for a test of the no - hair theorem , to crit 30 for any binary with mass ratio greater than 1.25 . a significant fraction of lisa events should satisfy this criterion ( see for example ) . in , the authors used numerical - relativity simulations of nonspinning black - hole binaries , with a variety of mass - ratios ranging from 1:1 to 1:11 , to compute the amplitude to which several different ringdown modes were excited , and hence an estimate of the snr with which lisa would be able to observe them . for all the mass ratios considered , the ( 3 , 3 ) mode was found to radiate between 2% and 20% of the energy of the ( 2 , 2 ) mode , and for mass ratios more unequal than 1:2 the ( 4 , 4 ) also radiated more than 1% of the energy of the ( 2 , 2 ) mode . for sources at a redshift of z = 1 , they estimated a total ringdown snr between 1000 and 20000 for black - hole masses in the range 1010 m and mass ratios from 1:1 to 1:25 , with individual snrs in the ( 2 , 2 ) , ( 2,1 ) , and ( 3 , 3 ) modes between several hundred and 10 000 . the mode snrs do not add in quadrature , but these snrs more than meet the requirements of for lisa to be able to carry out black - hole spectroscopy . the snrs realizable with elisa will be a few factors lower and the systems will tend to have lower masses , for which the intrinsic snrs are also smaller . therefore constraints from elisa will be weaker and it is possible that elisa will not be able to resolve sufficient separate modes , but this has not yet been explored . in , gossan et al . applied the results of to explore the practicality of using qnm radiation to test relativity . the authors considered mergers with mass ratios of 1:2 and constructed a waveform model comprised of four ringdown modes . they used bayesian methods and approached the problem of testing relativity in two ways : ( i ) determining the parameters of each ringdown mode separately and checking for consistency ; ( ii ) comparing the bayesian evidence of the gr model to a non - gr model constructed by allowing for deviations of the mode parameters from the gr predictions . the analysis was carried out for elisa and for the einstein telescope , the proposed third - generation ground - based detector . showed that method ( i ) could reveal inconsistencies of 1% in the qnm frequency for events of mass 10 m at a distance of 50 gpc and inconsistencies of 10% for systems of mass 10 m at 6 gpc . better constraints will come from systems at smaller distances and will therefore be observed with higher snr . using method ( ii ) , in the case of a signal from a 10 m black hole , 2% , 6% , and 10% deviations in the frequency of the dominant mode would be identifiable at distances of approximately 1 gpc , 5 gpc , and 8 gpc , respectively . deviations of 2% , 6% and 10% in the damping time of the dominant mode would only be detectable at distances of 200 mpc , 700 mpc , and 1.2 gpc , respectively . for a more massive system , of 10 m , a 2% deviation in the frequency / damping time of the dominant mode would be detectable out to 35/25 gpc , but deviations of 5% or more would be detectable at greater than 50 gpc . such massive systems are very rare , however , and the choice of a 1:2 mass ratio means that the qnm radiation is stronger than would be expected for typical elisa events , which are likely to have larger mass ratios . therefore this study was limited in extent , but these preliminary results suggest that qnms could place interesting constraints on gr modifications for the strongest signals detected by elisa . space - based qnm observations could be used directly to put constraints on alternative theories of gravity , but this will require a calculation of the qnms in those alternative theories . it was shown in that the equations governing black - hole perturbations in dynamical cs gravity were different from those in gr , with the consequence that the qnm spectrum would also be different . the authors did not compute qnms , but qnm frequencies for non - spinning black holes in dynamical cs gravity were computed in . the qnm spectrum was found to be different , due to the coupling of the black hole oscillations to the scalar field , but the detectability of these deviations by a gravitational - wave detector was not estimated . therefore , it is not yet clear at what level ringdown radiation will be able to constrain the cs coupling parameter . if a lisa - like observatory observes a mbh inspiral and does not detect qnm ringing from the merger remnant where radiation would be expected , this might indicate a violation of the cosmic - censorship hypothesis . qnm ringing arises as a result of gws becoming trapped near the horizon of the black hole . if a horizon was absent , for instance if a super - extremal ( a / m > 1 ) kerr metric was formed , we would not expect to observe the qnm radiation . this would be evidence for the existence of a naked singularity , and a counterexample to cosmic censorship , although not an indication of a problem with gr . although there are still many open questions , current research clearly indicates that low - frequency gw detectors will be able to make strong statements about the structure of the massive compact objects in the centers of galaxies by observing emris and by detecting ringdown radiation following supermassive black - hole mergers . at the very least , it will be possible to test that an observation is consistent with an inspiral into a kerr black hole , and to state quantitatively how so large a deviation from kerr could have been masked by instrumental noise . if a system differed significantly from kerr , we would identify the deviation and should be able to quantify its nature . how well we would be able to distinguish between different types of deviation ( e.g. , external matter vs. a different multipole structure of the central object ) is still not totally clear , but there are ongoing efforts in this direction . the tests of black - hole structure outlined in this section will rely on the detection of small differences between the observed gws and the gws expected in gr . these waveforms can be computed using black - hole perturbation theory , by evaluating the gravitational self - force , but despite extensive work in this area the calculation of the self - force is not yet complete even at first order in the mass ratio . the first - order gravitational self - force has been computed for circular and generic orbits in the schwarzschild spacetime and the scalar self - force is known for circular equatorial orbits in the kerr spacetime . recently , the first self - force - driven inspirals have been computed , under an adiabatic approximation for the gravitational self - force , and self - consistently for the scalar self - force .9 emri models will require knowledge of the gravitational self - force for generic orbits in kerr , which is still not available at first order . it has also been recognized that the radiative part of the second - order self - force will also be required to derive emri waveform phase to sufficient accuracy . a formalism for the evaluation of the second - order self - force has been developed , but not yet implemented . complete reviews of the current status of the self - force program can be found in [ 45 , 362 ] . additional complications in emri modeling arise from the need to accurately follow the transition of the inspiral through transient resonances . although many of these issues will have been resolved by the time data from a space - based gravitational - wave interferometer is available , they must be borne in mind in the interpretation of future results on tests of gr . low - frequency gw detectors will provide information on black - hole structure to a much higher precision than can be achieved by other techniques . it has been suggested that observations of imris with advanced ligo will have some power to do similar tests . this data will be available ten or more years prior to any space - based experiment . however , imri - based tests will be much weaker , since many fewer cycles of the gws will be detected in a single observation due to the larger mass ratio ( 10 10 ) ; furthermore , event rates are highly uncertain and accurate modeling of imris is far behind emri modeling [ 239 , 240 ] . an imri observation would be able to detect a deviation from kerr of fractional order unity in the quadrupole moment of a source , compared to the 10 fractional accuracies achievable with lisa emris . psaltis provides a thorough review of tests of strong - field gravity using electromagnetic observations . x - ray interferometers ( e.g. , the black hole imager ) will soon have the angular resolution to directly image the horizon of extragalactic black holes at distances of 1 mpc . the accretion flow for the milky way black hole , sgr a * , can already be imaged directly down to its innermost edge . however , interpretation of the observations is complicated by the need to simultaneously constrain the disc properties . observations at multiple wavelengths will be required to fit out the disc and directly image the shadow of the black hole on the disc.evidence for the existence of horizons also comes from observations of quiescent x - ray binaries . an x - ray binary typically comprises a star filling its roche lobe and transferring matter to a compact - object companion . that compact object can either be a neutron star or a black hole . if the object is a neutron star then most of the gravitational potential energy of the accreting matter has to be radiated away , but for black holes a significant amount of this energy can be advected through the black - hole horizon and is therefore not radiated to infinity . among systems in the same state of mass transfer , those containing neutron stars are expected to be systematically more luminous than those containing black holes , as has been observed in our galaxy.electromagnetic observations will really indicate the existence of a high - redshift surface in the system , and not necessarily an actual horizon . if a system contained a naked singularity with a high - redshift surface , but not a true event horizon , this would not be evident from the electromagnetic observations alone . by probing the multipole structure and verifying consistency with the no - hair theorem the highest - temperature emission from a disc comes from the innermost stable circular orbit ( isco ) , and the flux at that temperature is proportional to the isco radius squared . if the inferred spin was found to exceed one , this might indicate failure in the black - hole model . such spin determinations have typical errors of 10% , much greater than lisa s expected errors of 0.01% .indirect inference of the location of the inner edge of the accretion disc could also come from the interferometric observations mentioned above in the context of horizon detection . in the authors use radio interferometry to resolve the base of the jet in the galaxy m87 , finding it to be 5.5 0.4 schwarzschild radii . the jet - base radius is interpreted as being greater than or equal to the radius of the innermost edge , so this system provides evidence for prograde accretion onto a spinning black hole . observations of jets in other nearby galaxies may follow in the future , but the distance to which these structures could be resolved is quite small.accretion-disc mapping . particles in the accretion disc around a black hole move on circular geodesics of the metric . if the orbits in the disc could be mapped , this would allow spacetime mapping along the lines of ryan s theorem . two possible probes of accretion - disc structure have been identified : quasi - periodic oscillation ( qpo ) pairs and iron k lines . qpos have been used to constrain possible values of black - hole masses and spins , but there are uncertainties in the radius at which they originate , and in the resonance that gives rise to the pairs of lines . iron lines show broadening due to differential gravitational redshift and doppler shift at different points in the disc , but again their interpretation depends on unknown details of the disc geometry . in principle , the time variability of an iron line after a single flare event could constrain both the geometry and map the disc . future observations with instruments such as ixo / athena will have the time - resolution to attempt this . horizon detection . x - ray interferometers ( e.g. , the black hole imager ) will soon have the angular resolution to directly image the horizon of extragalactic black holes at distances of 1 mpc . the accretion flow for the milky way black hole , sgr a * , can already be imaged directly down to its innermost edge . however , interpretation of the observations is complicated by the need to simultaneously constrain the disc properties . observations at multiple wavelengths will be required to fit out the disc and directly image the shadow of the black hole on the disc . evidence for the existence of horizons also comes from observations of quiescent x - ray binaries . an x - ray binary typically comprises a star filling its roche lobe and transferring matter to a compact - object companion . that compact object can either be a neutron star or a black hole . if the object is a neutron star then most of the gravitational potential energy of the accreting matter has to be radiated away , but for black holes a significant amount of this energy can be advected through the black - hole horizon and is therefore not radiated to infinity . among systems in the same state of mass transfer , those containing neutron stars are expected to be systematically more luminous than those containing black holes , as has been observed in our galaxy . electromagnetic observations will really indicate the existence of a high - redshift surface in the system , and not necessarily an actual horizon . if a system contained a naked singularity with a high - redshift surface , but not a true event horizon , this would not be evident from the electromagnetic observations alone . by probing the multipole structure and verifying consistency with the no - hair theorem the highest - temperature emission from a disc comes from the innermost stable circular orbit ( isco ) , and the flux at that temperature is proportional to the isco radius squared . if the inferred spin was found to exceed one , this might indicate failure in the black - hole model . such spin determinations have typical errors of 10% , much greater than lisa s expected errors of 0.01% . indirect inference of the location of the inner edge of the accretion disc could also come from the interferometric observations mentioned above in the context of horizon detection . in the authors use radio interferometry to resolve the base of the jet in the galaxy m87 , finding it to be 5.5 0.4 schwarzschild radii . the jet - base radius is interpreted as being greater than or equal to the radius of the innermost edge , so this system provides evidence for prograde accretion onto a spinning black hole . observations of jets in other nearby galaxies may follow in the future , but the distance to which these structures could be resolved is quite small . particles in the accretion disc around a black hole move on circular geodesics of the metric . if the orbits in the disc could be mapped , this would allow spacetime mapping along the lines of ryan s theorem . two possible probes of accretion - disc structure have been identified : quasi - periodic oscillation ( qpo ) pairs and iron k lines . qpos have been used to constrain possible values of black - hole masses and spins , but there are uncertainties in the radius at which they originate , and in the resonance that gives rise to the pairs of lines . iron lines show broadening due to differential gravitational redshift and doppler shift at different points in the disc , but again their interpretation depends on unknown details of the disc geometry . in principle , the time variability of an iron line after a single flare event could constrain both the geometry and map the disc . future observations with instruments such as ixo / athena will have the time - resolution to attempt this . electromagnetic observations are generally hampered by a lack of knowledge of the physics of the material that is generating the radiation . gw systems are , by contrast , very clean since the same theory describes the spacetime and the radiation generation . while there is some hope that future electromagnetic observations will perform crude spacetime mapping , lisa emri observations will improve any previous constraints by orders of magnitude . although the bibliography of this review lists more than 500 references , fundamental tests of gravity remain the least explored sector of gw astronomy . it is also one where new discoveries may be hardest to come by even if they would be momentous if they do . space - based gravitational - wave detectors will improve certain existing constraints on alternative theories ; even more important , they will provide novel and unique opportunities to precisely characterize the poorly explored nonlinear , dynamical sector of gravitation , as well as the properties of gravitational radiation fields . these measurements will provide a definite confirmation that einstein s theory , born from insight and beauty , applies in the most extreme regimes ; or they may expose new phenomena , leading to new models for gravitational physics . the research that we have reviewed spans a broad assortment of gw observations , which probe many disparate aspects of gravitational phenomenology . some of these investigations have yet to be refined to the same level of formal rigor as other , more astrophysical applications of gw astronomy . nevertheless , they paint an exciting picture of the expected fundamental - physics payoff of space - based detectors . we expect that this will remain an active and intriguing research area for many years : thus , it is appropriate that this review should be living , and we welcome the suggestions of our readers in improving it and keeping it up to date .

we review the tests of general relativity that will become possible with space - based gravitational - wave detectors operating in the 105 1 hz low - frequency band . the fundamental aspects of gravitation that can be tested include the presence of additional gravitational fields other than the metric ; the number and tensorial nature of gravitational - wave polarization states ; the velocity of propagation of gravitational waves ; the binding energy and gravitational - wave radiation of binaries , and therefore the time evolution of binary inspirals ; the strength and shape of the waves emitted from binary mergers and ringdowns ; the true nature of astrophysical black holes ; and much more . the strength of this science alone calls for the swift implementation of a space - based detector ; the remarkable richness of astrophysics , astronomy , and cosmology in the low - frequency gravitational - wave band make the case even stronger .

Introduction The Theory of Gravitation Space-Based Missions to Detect Gravitational Waves Summary of Low-Frequency Gravitational-Wave Sources Gravitational-Wave Tests of Gravitational Physics Tests of the Nature and Structure of Black Holes Discussion

PMC4553273

the local anesthetic ( 1% lidocaine with epinephrine , 1:100,000 for tendon surgery in 1 or 2 digits or in the palm ) is injected to the digits of the patient in the waiting area starting 30 minutes before surgery for anesthesia . injection of such mixture to the hand was found to be safe.123456 ) waiting after injection allows the epinephrine to take effect and provide an adequately dry working field . 1 ) . we keep the total dose of infiltration less than 7 mg / kg . usually , less than 50 ml is required for tendon surgery in the hand ( including digits and palm areas ) , and we use premixed 1% lidocaine with 1:100,000 epinephrine . occasionally , 50 to 100 ml is required when surgery is involved in 3 or 4 digits or in the forearm ; in these cases , we dilute with saline to a concentration of 0.5% lidocaine with 1:200,000 epinephrine . the injection starts proximally with a fine needle ( 25 or 27 gauge ) ( fig . is injected in the most proximal part of the likely dissection area to block the nerves distally . this site is located in the distal part of the palm for a zone 2 tendon repair . in this way , the large volume of the first injection will bathe all of the nerves leading to the area of the next injections . for a zone 1 tendon repair , we usually inject the solution similarly as for a zone 2 tendon repair . in making the first injection , we insert the needle perpendicular to the skin into the subcutaneous fat to reduce pain of injection.6 ) then the syringe is stabilized with fingers propped on the skin to avoid needle wobble . wait 15 to 45 seconds until the patient tells us that the sting is gone . then , we proceed to inject the rest of infiltration very slowly ( more than 5 minutes ) without moving the needle ( fig . sometimes , i make this first injection in a quicker fashion than i described above and inject continuously in the way that we all use for local anesthesia , if the patient tolerates well . about 10 to 15 minutes after the first injection , the whole distal area of dissection is almost or totally numb . the next three injections are used mainly for the epinephrine vasoconstriction effect . between both digital nerves , 2 ml of infiltration is injected subcutaneously at the palmar digital crease , so the bevel of the needle does not lacerate the nerve fascicles ( fig . another 2 ml of infiltration is also injected subcutaneously at the digital crease of the proximal interphalangeal ( pip ) joint between both digital nerves ( fig . the final 1 ml of infiltration is then injected subcutaneously at the digital crease of the distal interphalangeal ( dip ) joint between both digital nerves ( fig . the surgery starts 30 minutes after the first injection or 15 minutes after completion of all injections . the sheath is opened through a small incision along the midline , about 1 to 1.5 cm usually . retraction of the tendon ends , especially the proximal tendon end , is very common . flexion of the distal finger joints helps bring the distal end into the operating field . if the tendon is cut in zone 2 , flexion of the metacarpophalangeal ( mp ) joint sometimes helps the proximal stump advance to the sheath opening area , but very often a separate incision at the distal palm is needed to find the proximal stump and it is advanced distally through the preserved sheath to approximate the distal tendon . if the tendon is cut in zone 1 , flexion of the pip joint usually brings the proximal end back to the operative field . during the surgery , some mild bleeding may be seen when the skin incision is made , but after wiping the sites with gauzes , the bleeding usually stops . we no longer use cautery but occasionally use a hemostat on bigger vessels for a few minutes to achieve hemostasis . the flexor digitorum profundus ( fdp ) tendon is repaired with a 6-strand core suture repair ( 4 - 0 suture , m - tang method using looped suture ( fig . alexandria , va , usa ) , or a triple kessler method with asymmetric core suture purchases at two tendon stumps8 ) ) plus a simple running peripheral suture ( 6 - 0 suture ) ( fig . core suture purchases should be at least 7 mm , ideally around 10 cm ( fig . 5 ) . this is a very important requirement for the core suture.910 ) the core suture is performed with some extent of tension to make the two tendon ends approximate tightly to increase gapping resistance , but not too tight to avoid marked tendon bulkiness . it is important to make the repair a bit tensed , rather than being loose or tension - free . my method is to tension the core suture lines to the degree that makes the tendon segments within the core sutures shortened slightly , by approximately 10% ( fig . 5).1112 ) the lacerated flexor digitorum superficialis ( fds ) tendon is repaired when the wound is clean and the repair is easy and the cut is not in the area of zone under the a2 pulley . in many cases , i do not repair the fds tendon , especially when at delayed primary repair , and when in the a2 pulley area , or when fds tendon retracted proximally . repair of damaged synovial sheath is usually not necessary and whether the sheath is closed does not affect outcomes . however , it is necessary to take care to preserve most or most parts of the annular pulleys if possible . when other annular pulleys are intact , we can vent the a4 pulley entirely if the tendon ends can pass under this narrow pulley or the pulley restricts tendon gliding ( which is often the case).7 ) if the injury site is at or around the a2 pulley , a part ( 1/2 to 2/3 ) of the a2 pulley can be vented.6 ) my method of venting is a direct cut along the volar midline using scissors . pulley venting may be done together with cutting of a part of adjacent synovial sheath . the total length of sheath - pulley release in the site of repair surgery is 1.5 cm or 2 cm depending on the finger length.6 ) our studies show that this length of release allows ample tendon gliding but will not cause tendon bowstringing when other sheath parts are intact . i have performed such venting for over 10 years and have not seen functional damage to the finger after properly doing the venting . we ask the patient to actively extend and flex the surgically repaired digits . this test verifies that the repair is strong and is able to tolerate early active digital motion during rehabilitation.1314 ) this test is called " digital extension - flexion test."14 ) there are thee needed parts of this test : ( 1 ) full active extension to verify no gapping between the tendon ends ( fig . 6 ) ; ( 2 ) smooth active flexion to verify smooth gliding of the tendon and its repair site ; and ( 3 ) active flexion to almost totally flex the digit to verify that the no pulley prevents tendon gliding . if the examination creates a visible gap between the two tendon ends during finger extension , the suture is usually not tight enough and needs to be replaced with a tighter suture with proper tension to avoid rupture . here , i strongly stress that the repeated total active movement examination may be needed to verify that the repair is strong and pulley release is sufficient . the allowable length of the pulley release for the a2 is 2/3 , and the entire a4 pulley can be vented with a part of the synovial sheath when all other annular pulleys are well preserved . usually this venting allows ample tendon gliding but will not cause tendon bowstringing when other sheath parts are intact . repair of flexor tendon in proximal zone 1 can use similar approaches because only fdp tendon is repaired . to find or pass the retracted proximal end , the a4 pulley may need entire venting if the repair is very close to that pulley . a separate incision in the proximal part of the digit or distal palm may be necessary to find the retracted fdp tendon and a4 may need total venting to pass the proximal stump distally . the patient is protected with a very short forearm based splint , from about 6 - 8 cm above the wrist to the tip of the finger , with the wrist at 20 to 30 flexion , mp joint at slight flexion , and the interphalangeal joints in extension . 15 ) i sometimes put the patient 's wrist in neutral position.715 ) i think , with a strong repair method and validation of good repair using the intraoperative extension - flexion test , we have a greater room to place the wrist at a position between a wrist neutral position and slight flexion , and may even slight extension . the early active motion that i use is a combined passive - active motion , starting from 3 to 5 days after the surgery.15 ) i do not move the operated digits in the first 3 days after surgery because edema is prominent , pain is severe , and adhesions do not form in this period . motion starts from day 4 or 5 . in the initial 1 to 2 weeks after surgery , we keep the active finger flexion within only one - third of the total range of finger motion ( fig . the partial range of active motion is progressively increased to two - thirds in week 3 or 4 after surgery . the patient should avoid full range of active flexion in the first 2 to 3 weeks after surgery because the final extreme digital flexion produces the greatest bending force to the tendon and tendon is prone to disruption . i sometimes start full active motion even a little later in the patient who had tendon repairs in multiple fingers , or when the tendon is repaired 3 to 5 weeks after trauma or after repair of ruptured primary repair . in performing these motion exercises , the full range of passive digital motion ( 10 to 30 repetitions ) the number of exercise sessions can range from 5 to 6 times per day and hourly in daytime , depending on the need of motion to prevent or correct digital joint stiffness and patient 's desire or availability for the exercise . four to 5 sessions are minimal each day and 10 to 30 repetitions of passive motion and 20 to 30 repetitions of active motion are minimal for each session in our practice . i do not order patients to move hourly , but some patients prefer to do exercise whenever he or she has free time . i encourage full active extension of the interphalangeal joints in all the postsurgical weeks and encourage differential pip and dip joint motions . many patients need further extension exercises because of some extension or flexion lags , which can be corrected in subsequent exercises . 8) , and persistent exercises of up to 5 or 6 month may still show improvement of function . the local anesthetic ( 1% lidocaine with epinephrine , 1:100,000 for tendon surgery in 1 or 2 digits or in the palm ) is injected to the digits of the patient in the waiting area starting 30 minutes before surgery for anesthesia . injection of such mixture to the hand was found to be safe.123456 ) waiting after injection allows the epinephrine to take effect and provide an adequately dry working field . 1 ) . we keep the total dose of infiltration less than 7 mg / kg . usually , less than 50 ml is required for tendon surgery in the hand ( including digits and palm areas ) , and we use premixed 1% lidocaine with 1:100,000 epinephrine . occasionally , 50 to 100 ml is required when surgery is involved in 3 or 4 digits or in the forearm ; in these cases , we dilute with saline to a concentration of 0.5% lidocaine with 1:200,000 epinephrine . the injection starts proximally with a fine needle ( 25 or 27 gauge ) ( fig . is injected in the most proximal part of the likely dissection area to block the nerves distally . this site is located in the distal part of the palm for a zone 2 tendon repair . in this way , the large volume of the first injection will bathe all of the nerves leading to the area of the next injections . for a zone 1 tendon repair , we usually inject the solution similarly as for a zone 2 tendon repair . in making the first injection , we insert the needle perpendicular to the skin into the subcutaneous fat to reduce pain of injection.6 ) then the syringe is stabilized with fingers propped on the skin to avoid needle wobble . wait 15 to 45 seconds until the patient tells us that the sting is gone . then , we proceed to inject the rest of infiltration very slowly ( more than 5 minutes ) without moving the needle ( fig . sometimes , i make this first injection in a quicker fashion than i described above and inject continuously in the way that we all use for local anesthesia , if the patient tolerates well . about 10 to 15 minutes after the first injection , the whole distal area of dissection is almost or totally numb . the next three injections are used mainly for the epinephrine vasoconstriction effect . between both digital nerves , 2 ml of infiltration is injected subcutaneously at the palmar digital crease , so the bevel of the needle does not lacerate the nerve fascicles ( fig . another 2 ml of infiltration is also injected subcutaneously at the digital crease of the proximal interphalangeal ( pip ) joint between both digital nerves ( fig . the final 1 ml of infiltration is then injected subcutaneously at the digital crease of the distal interphalangeal ( dip ) joint between both digital nerves ( fig . the surgery starts 30 minutes after the first injection or 15 minutes after completion of all injections . the sheath is opened through a small incision along the midline , about 1 to 1.5 cm usually . retraction of the tendon ends , especially the proximal tendon end , is very common . flexion of the distal finger joints helps bring the distal end into the operating field . if the tendon is cut in zone 2 , flexion of the metacarpophalangeal ( mp ) joint sometimes helps the proximal stump advance to the sheath opening area , but very often a separate incision at the distal palm is needed to find the proximal stump and it is advanced distally through the preserved sheath to approximate the distal tendon . if the tendon is cut in zone 1 , flexion of the pip joint usually brings the proximal end back to the operative field . during the surgery , some mild bleeding may be seen when the skin incision is made , but after wiping the sites with gauzes , the bleeding usually stops . we no longer use cautery but occasionally use a hemostat on bigger vessels for a few minutes to achieve hemostasis . the flexor digitorum profundus ( fdp ) tendon is repaired with a 6-strand core suture repair ( 4 - 0 suture , m - tang method using looped suture ( fig . alexandria , va , usa ) , or a triple kessler method with asymmetric core suture purchases at two tendon stumps8 ) ) plus a simple running peripheral suture ( 6 - 0 suture ) ( fig . 4 ) . core suture purchases should be at least 7 mm , ideally around 10 cm ( fig . this is a very important requirement for the core suture.910 ) the core suture is performed with some extent of tension to make the two tendon ends approximate tightly to increase gapping resistance , but not too tight to avoid marked tendon bulkiness . it is important to make the repair a bit tensed , rather than being loose or tension - free . my method is to tension the core suture lines to the degree that makes the tendon segments within the core sutures shortened slightly , by approximately 10% ( fig . 5).1112 ) the lacerated flexor digitorum superficialis ( fds ) tendon is repaired when the wound is clean and the repair is easy and the cut is not in the area of zone under the a2 pulley . in many cases , i do not repair the fds tendon , especially when at delayed primary repair , and when in the a2 pulley area , or when fds tendon retracted proximally . repair of damaged synovial sheath is usually not necessary and whether the sheath is closed does not affect outcomes . however , it is necessary to take care to preserve most or most parts of the annular pulleys if possible . when other annular pulleys are intact , we can vent the a4 pulley entirely if the tendon ends can pass under this narrow pulley or the pulley restricts tendon gliding ( which is often the case).7 ) if the injury site is at or around the a2 pulley , a part ( 1/2 to 2/3 ) of the a2 pulley can be vented.6 ) my method of venting is a direct cut along the volar midline using scissors . pulley venting may be done together with cutting of a part of adjacent synovial sheath . the total length of sheath - pulley release in the site of repair surgery is 1.5 cm or 2 cm depending on the finger length.6 ) our studies show that this length of release allows ample tendon gliding but will not cause tendon bowstringing when other sheath parts are intact . i have performed such venting for over 10 years and have not seen functional damage to the finger after properly doing the venting . we ask the patient to actively extend and flex the surgically repaired digits . this test verifies that the repair is strong and is able to tolerate early active digital motion during rehabilitation.1314 ) this test is called " digital extension - flexion test."14 ) there are thee needed parts of this test : ( 1 ) full active extension to verify no gapping between the tendon ends ( fig . 6 ) ; ( 2 ) smooth active flexion to verify smooth gliding of the tendon and its repair site ; and ( 3 ) active flexion to almost totally flex the digit to verify that the no pulley prevents tendon gliding . if the examination creates a visible gap between the two tendon ends during finger extension , the suture is usually not tight enough and needs to be replaced with a tighter suture with proper tension to avoid rupture . here , i strongly stress that the repeated total active movement examination may be needed to verify that the repair is strong and pulley release is sufficient . the allowable length of the pulley release for the a2 is 2/3 , and the entire a4 pulley can be vented with a part of the synovial sheath when all other annular pulleys are well preserved . usually this venting allows ample tendon gliding but will not cause tendon bowstringing when other sheath parts are intact . repair of flexor tendon in proximal zone 1 can use similar approaches because only fdp tendon is repaired . to find or pass the retracted proximal end , the a4 pulley may need entire venting if the repair is very close to that pulley . a separate incision in the proximal part of the digit or distal palm may be necessary to find the retracted fdp tendon and a4 may need total venting to pass the proximal stump distally . the patient is protected with a very short forearm based splint , from about 6 - 8 cm above the wrist to the tip of the finger , with the wrist at 20 to 30 flexion , mp joint at slight flexion , and the interphalangeal joints in extension . 15 ) i sometimes put the patient 's wrist in neutral position.715 ) i think , with a strong repair method and validation of good repair using the intraoperative extension - flexion test , we have a greater room to place the wrist at a position between a wrist neutral position and slight flexion , and may even slight extension . the early active motion that i use is a combined passive - active motion , starting from 3 to 5 days after the surgery.15 ) i do not move the operated digits in the first 3 days after surgery because edema is prominent , pain is severe , and adhesions do not form in this period . motion starts from day 4 or 5 . in the initial 1 to 2 weeks after surgery , we keep the active finger flexion within only one - third of the total range of finger motion ( fig . 7 ) . the partial range of active motion is progressively increased to two - thirds in week 3 or 4 after surgery . the patient should avoid full range of active flexion in the first 2 to 3 weeks after surgery because the final extreme digital flexion produces the greatest bending force to the tendon and tendon is prone to disruption . i sometimes start full active motion even a little later in the patient who had tendon repairs in multiple fingers , or when the tendon is repaired 3 to 5 weeks after trauma or after repair of ruptured primary repair . in performing these motion exercises , the full range of passive digital motion ( 10 to 30 repetitions ) the number of exercise sessions can range from 5 to 6 times per day and hourly in daytime , depending on the need of motion to prevent or correct digital joint stiffness and patient 's desire or availability for the exercise . four to 5 sessions are minimal each day and 10 to 30 repetitions of passive motion and 20 to 30 repetitions of active motion are minimal for each session in our practice . i do not order patients to move hourly , but some patients prefer to do exercise whenever he or she has free time . i encourage full active extension of the interphalangeal joints in all the postsurgical weeks and encourage differential pip and dip joint motions . many patients need further extension exercises because of some extension or flexion lags , which can be corrected in subsequent exercises . 8) , and persistent exercises of up to 5 or 6 month may still show improvement of function . therefore , the patient can actively move the digits or the hand to ensure that tenolysis is adequate and the tendon is strong enough to move the tendon . the method of anesthesia is the same as we described earlier for primary repair , and surgical incisions for tenolysis are the same with those used traditionally . the only difference is that the surgeons can ask the patient to actively flex or extend the digit or the hand to see active gliding of the tendon . if the tenolysis is sufficient , the surgery is complete . if the tenolysis is insufficient , as indicated by insufficient digit or hand motion , further release of scar around the tendon is necessary such active motions help the surgeon to test the strength of these tendons . if the tendon is remarkably elongated or is broken when the digits or the hand actively moved , tendon reconstruction using a tendon grafting should be considered . during active motion , strength of the pulley can be tested as well and whether the pulleys restrict tendon gliding can be assessed . if the pulleys are broken during active tendon motion , important annular pulleys may need reconstruction . restriction of the tendon gliding from the pulleys may need further release of adhesions or a pulley plasty or pulley shortening procedure . tendon transfer is actually best indicated for such wide - awake surgery , because adjusting tension of the transfer has always been difficult . with the patient being awake , the digits or the hand can move actively to determine correct tension of the transfer . the patient should be asked to perform the desired action after transferring the tendon before the surgeon places the suture to fix the transferred tendon to the damaged tendon . this practice has been a routine for some hand surgeons now , and has been used in most commonly performed tendon transfers such as extensor indicis proprius transfer to the extensor pollicis longus , flexor carpi radialis transfer to the extensor digitorum communis . the anesthesia method is the same as described for the flexor tendon repair in the palm . if a lengthy incision or dissection over an extensive area is necessary in the forearm , 0.5% lidocaine with 1:200,000 epinephrine is used to decrease the total amount of injected epinephrine . strength and gliding of the tendon suture site ( usually pulvertaft weave suture ) are also possible with the patient actively moving the digits or the hand . besides tendon surgery , similar wide - awake surgery has been used in carpal tunnel release , cubital tunnel release , wrist arthroscopy , and some benign tumor resection.16171819 ) these uses have greatly reduced the cost of the surgery and diminished the use of supplies during surgery.16171819 ) many patients now can be operated in a minor procedure room . in our department , we established a wide - awake surgical room right in the ward . many patients do not need to go to the main operating theatre to have surgery . such a wide - wake surgical room reduces surgical waiting time and save the time of patient transfer and recovery , representing a huge increase in efficiency of surgical treatment as compared to previous surgical settings .

tendon surgery is unique because it should ensure tendon gliding after surgery . tendon surgery now can be performed under local anesthesia without tourniquet , by injecting epinephrine mixed with lidocaine , to achieve vasoconstriction in the area of surgery . this method allows the tendon to move actively during surgery to test tendon function intraoperatively and to ensure the tendon is properly repaired before leaving the operating table . i applied this method to primary flexor tendon repair in zone 1 or 2 , tenolysis , and tendon transfer , and found this approach makes tendon surgery easier and more reliable . this article describes the method that i have used for tendon surgery .

PRIMARY FLEXOR TENDON REPAIR IN ZONE 1 OR 2 Anesthetic Solution Methods of Injection Surgical Procedures Extension-Flexion Test Postoperative Care TENOLYSIS TENDON TRANSFER

PMC3228652

the vision and change deliberations on transforming undergraduate biology education recently articulated a need to engage students in the process of science and to present science as a vibrant and active field ( american association for the advancement of science , 2010 ) . extensive pedagogic research concludes that participation in open - ended research endeavors fosters a sense of ownership over a biological subject , and enhances teaching and learning in biological sciences ( teagle foundation , 2007 ) . developing innovative cross - disciplinary approaches and empowering faculty with the tools to implement novel strategies remains a challenge at all levels of undergraduate education . in the last 5 yr , the rise of next - generation ( nextgen ) sequencing approaches in addressing biological problems has been spectacular , but incorporating nextgen sequencing data for active teaching in the undergraduate curriculum remains a major challenge . for faculty at small and medium - sized institutions of higher education , high teaching loads , lack of access to state - of - the - art equipment , and budgetary constraints typically conspire to inhibit faculty from considering nextgen sequencing in their own experiments . high capital costs , extraordinarily high rates of technological change , and daunting computational and analytical requirements make the technology exceptionally challenging to assimilate into the undergraduate curriculum . the genome consortium for active teaching ( gcat ; campbell et al . , 2006 ) was developed a decade ago to meet similar challenges in relation to the use of microarrays in undergraduate biology education . gcat offers highly discounted microarray chips and array scanning and a supporting network of faculty expertise to educators working with undergraduates . in one decade , the effort has trained over 360 faculty and 24,000 undergraduates in the use and interpretation of microarray data . the newly trained students were enrolled in primarily undergraduate institutions , including those that historically serve underrepresented populations . gcat has met many of the goals of the bio2010 report ( campbell et al . , 2007 ) , and recently expanded its focus into synthetic biology ( wolyniak et al . , 2010 ) . now that nextgen sequencing is rapidly superseding microarray technology for a variety of technical and economic considerations , gcat and others recognized the need to find cost - effective and innovative strategies to facilitate active teaching of nextgen technology at the undergraduate level . understanding the advantages and limitations of continually evolving transformative technologies like nextgen sequencing is essential preparation for future life scientists , medical professionals , and , indeed , a scientifically literate citizenry , as the age of personalized medicine moves toward becoming reality . in addition , analyzing raw sequence data provides students with learning opportunities that underscore interdisciplinary concepts central and relevant to studies of all forms of life . in this paper , we report proceedings of a workshop from a national science foundation ( nsf)funded incubator grant for research coordination networks for undergraduate biology education ( rcn / ube ) . the network aimed to collaborate with a centrally located genome - sequencing core facility at the pennsylvania state university ( psu ) to enable undergraduate students and faculty in the mid - atlantic region to access state - of - the - art sequencing technology . initial network participants included juniata college , susquehanna university , duquesne university , hampton university , morgan state university , ramapo college of new jersey , gettysburg college , lycoming college , lock haven university , mount aloysius college , bucknell university , and hood college , with the genome - sequencing facility at psu supporting the data acquisition and dissemination aspects of the initiative . the meeting was held july 1114 , 2011 , at juniata college and psu and included the individuals who helped write the incubator grant and invited speakers malcolm campbell ( davidson college ) , anton nekrutenko ( psu ) , istvan albert ( psu ) , and bill morgan ( college of wooster ) . through presentations and periodic whole - group discussions , we worked together to exchange ideas to develop a structure to approach the problem of introducing nextgen sequencing to undergraduates . during the course of the meeting , a number of parallels emerged between the thinking of the participants and the philosophy of gcat . members of both groups valued the academic freedom provided by their ability to choose and direct their own research and scholarly activities . both groups recognized the value of communal support from colleagues at similar small institutions to help compensate for lack of a critical mass of peers on each campus . both groups recognized the strategic need to partner with other groups , like the microarray manufacturers in gcat 's initial plan , or genome - sequencing facilities , such as psu . all of these considerations suggested that the mid - atlantic network for nextgen sequencing could operate more effectively and enable the approach to be replicated elsewhere if it partnered with gcat as a new arm of that consortium . gcat has established an efficient dissemination strategy through its website and listserv , and many of the members currently using microarrays will be poised to transition to nextgen sequencing as it replaces gene chip technology . our shared values and the success of the rcn / ube grant led to an agreement with malcolm campbell for our rcn / ube to become gcat - seek(quence ) and to complement another gcat initiative in the emerging field of synthetic biology ( gcat - synbio ; wolyniak et al . , 2010 ) . at our network meeting , we formed a nascent community of biologists from distinct areas ( e.g. , molecular biology , environmental science , plant biology , microbiology ) aiming to develop parallel research studies in the scholarship of teaching and learning and discovery science . the specific goals of the workshop were to : 1 ) learn lessons from the gcat model ; 2 ) learn the scope of nextgen sequencing technology , applications , and analysis ; 3 ) develop common learning goals for students using this technology and develop appropriate methods of assessment and ; 4 ) develop goals and an administration plan for the network . malcolm campbell presented the keynote address on gcat , describing lessons learned from administration of the consortium . in particular , he emphasized the importance of an undergraduate focus , inclusion of minority - serving institutions , assessment of educational activities , advertisement of the network , faculty development , and taking on the most difficult problems related to a technology to make the network valuable . the gcat model also recognized the importance of the investigator retaining ownership in the direction of his or her research . this was one of the key reasons for adopting a model in which an investigator requested the raw sequence data related to their research expertise and passion . deb grove and craig praul ( codirectors of the psu genome core facility ) detailed the latest sequencing technologies , applications , and costs associated with their ion torrent pgm , roche 454flx , and solid 5500xl platforms . these are massively parallel dna - sequencing machines capable of providing hundreds of millions to tens of billions of nucleotides of dna sequence data in about a week . the resulting data must be processed using specialized bioinformatics techniques that may require high - powered computers . it was determined that up to 50% of costs could be cut when individual researchers cooperate to share sequencing runs . unique dna sequence adapters ( bar codes ) may be ligated onto an investigator 's dna fragments before sequencing . this process allows each investigator 's samples to be individually labeled , pooled with other samples , and automatically separated after bulk sequencing . psu biochemistry and molecular biology faculty anton nekrutenko and istvan albert framed challenges and approaches to nextgen data analysis . anton nekrutenko described the galaxy bioinformatics analysis framework that he and his colleagues developed ( blankenberg et al . , 2010 ; he emphasized the importance of the evolutionary underpinnings of bioinformatics analysis , and how comparison is key to understanding genomes . istvan albert , director of the bioinformatics consulting center at psu , suggested that bioinformatics analysis is challenging , because it is a highly interdisciplinary science incorporating information technology to manage data , computer science to analyze data , statistics to find meaningful patterns , and biology to form relevant hypotheses . he stressed that bioinformatics can not be learned passively from a book but requires active - learning approaches and student commitment . the challenge facing undergraduate faculty in introducing bioinformatics was addressed by ash stuart ( ramapo college of new jersey ) , eric sakk ( morgan state university ) , and bill morgan ( college of wooster ) , who is working on a related initiative in genomics education . the effectiveness of interdisciplinary approaches , open - ended inquiry , case studies , online student learning communities , undergraduate conferences , exchange of students between schools , and interaction among students in different disciplines on a single campus all had the potential to improve student communication , collaboration , and leadership skills . it was stressed that one of the desired skills students should acquire was adaptability , because the field of bioinformatics changes so rapidly . having a forum to discuss the impact of genomic sciences and bioinformatics analysis using examples from the daily news bill morgan discussed his progress toward a free , online genomics textbook focused on interactive case studies with mathematical sidebars and modeled after the text , discovering genomics , proteomics , and bioinformatics , by campbell and heyer ( 2006 ) . he has assembled a group of faculty members from throughout the united states with expertise in genomics to help coordinate the development of learning modules in 10 topic areas . the workshop participants were particularly interested in reviewing the learning objectives for the genome - sequencing topic . it was suggested that a template with learning objectives , protocols , and assessment instruments be developed for the next - generation sequencing module that would encourge faculty adoption because it would allow customization of its activities to the datasets of individual investigators . no innovation in education can be considered successful if it is not subjected to rigorous evaluation and assessment in the context of defined learning objectives . the workshop participants discussed the learning objectives the network should have , and formed an assessment leadership team chaired by tammy tobin ( susquehanna university ) and jay hosler ( juniata college ) . this team will guide development of appropriate instruments to monitor student outcomes for network participants , as well as to support individual faculty in assessing the impact of students working with raw sequence data in individual classes . core learning outcomes proposed for the gcat - seek network were the ability for instructors and students to do the following : explain each step in the generation and analysis of nextgen sequence data.discuss the basic biology assumptions that underlie sequence analysis ( e.g. , evolution , structure , and function).evaluate the strengths and weaknesses of the methods used in nextgen sequencing , including the impact that data quality has on bioinformatics analysis.construct a testable hypothesis and experimental design that uses nextgen sequencing and bioinformatics tools.choose and justify the appropriate methods for a specific nextgen sequencing application . discuss the basic biology assumptions that underlie sequence analysis ( e.g. , evolution , structure , and function ) . evaluate the strengths and weaknesses of the methods used in nextgen sequencing , including the impact that data quality has on bioinformatics analysis . construct a testable hypothesis and experimental design that uses nextgen sequencing and bioinformatics tools the goal is to have best teaching practices established and validated through appropriate assessment and distributed to all of the network participants and their colleagues . following whole - group discussion , it was determined the agreed purpose of gcat - seek is to 1 ) bring functional genomic methods into the undergraduate curriculum , primarily through independent and classroom - based student research using centralized core facilities to make nextgen sequence data accessible to undergraduates ; 2 ) create a clearinghouse of information for educators to use when teaching nextgen sequencing and related topics ; 3 ) create a large database of raw data and analyzed results for pedagogical use by gcat - seek members ; and 4 ) develop a global network of educators who are using functional genomics and nextgen sequencing in the undergraduate curriculum . gcat - seek specifically aims to obtain group discounts at regional research - intensive core facilities , to negotiate software discounts , and to garner support for mini - grants to help cover the cost of the initial sequencing runs for network participants . the network aims to support its members through online listservs , periodic workshops , and meetings , following an approach similar to that successfully used by other gcat groups ( campbell et al . , 2006 ) . the network approach will add efficiency by coordinating projects and partnering investigators with appropriate sequencing platforms . given that even the smallest purchasable unit of nextgen sequence will often contain a great excess of information for any given project and that many projects can be combined using bar codes ( as discussed in the introductory paragraph ) , an organized staging for related samples from different investigators was envisioned . this may reduce cost of data acquisition to a few thousand dollars from departmental budgets or mini - grant programs . furthermore , additional cost efficiency for network participants can be achieved through coordination of the synthesis and maintenance of a database of highly purified bar - coded primers for metagenomic analysis . at a time of severe budget scrutiny , the efficiency and cost - effectiveness of the proposed approach is apparent . sequencing cores are not running at capacity , and technological advances are lowering sequencing costs . bioinformatics programs , databases , and computing requirements for many types of projects are all either already in the public domain or well within the budgets of even the smallest undergraduate colleges . given sufficient interest , regional replication of some elements of gcat - seek in the future should be considered as a means of lowering travel costs for meetings and workshops for participants and allowing students to more easily visit genome core facilities . thus , with a modest investment , this program can start to meet the challenges of training the next generation of life scientists by engaging undergraduates in the process of science presented in the context of modern technology .

to transform undergraduate biology education , faculty need to provide opportunities for students to engage in the process of science . the rise of research approaches using next - generation ( nextgen ) sequencing has been impressive , but incorporation of such approaches into the undergraduate curriculum remains a major challenge . in this paper , we report proceedings of a national science foundation funded workshop held july 1114 , 2011 , at juniata college . the purpose of the workshop was to develop a regional research coordination network for undergraduate biology education ( rcn / ube ) . the network is collaborating with a genome - sequencing core facility located at pennsylvania state university ( university park ) to enable undergraduate students and faculty at small colleges to access state - of - the - art sequencing technology . we aim to create a database of references , protocols , and raw data related to nextgen sequencing , and to find innovative ways to reduce costs related to sequencing and bioinformatics analysis . it was agreed that our regional network for nextgen sequencing could operate more effectively if it were partnered with the genome consortium for active teaching ( gcat ) as a new arm of that consortium , entitled gcat - seek(quence ) . this step would also permit the approach to be replicated elsewhere .

THE CHALLENGE OF TRANSFORMING UNDERGRADUATE EDUCATION THE MEETING GCAT-SEEK: VISION AND APPROACH WHAT'S NEXT?

PMC4327299

this report evaluated the efficacy of three brushes and one biofilm disclosing agent in complete denture cleansing . methods : twenty - seven wearers of maxillary dentures were distributed into three groups and received different brushes : oral b40 , conventional toothbrush ( oral b ) ; denture , denture - specific brush ( condor ) ; johnson & johnson , denture - specific brush ( johnson & johnson ) . the 60-day experimental period was divided into two techniques : i - brushing ( brush associated with a paste - dentu creme , dentco ) three times a day ; ii - brushing and daily application of 1% neutral red on the denture internal surface . biofilm quantification was carried out weekly and the areas with dye biofilm were obtained by means of image tool 2.02 software . biofilm removal was more effective during technique ii ( wilcoxon test : p=0.01 ) for the three groups of brushes . when the brushes were compared in technique i , the kruskal wallis test indicated statistical difference between denture x johnson & johnson and denture x oral b40 , in which the denture was more efficient . for technique the disclosed application promoted more efficacy on biofilm removal , regardless of the brush used . although many reports evaluate the efficacy of denture cleansers , surveys point out that removal of denture biofilm by denture wearers is precarious13,5,6,23 . deficient patient orientation , inadequate divulgation of specific denture cleanser materials , intrinsic characteristics of prosthetic appliances and deficient manual dexterity of elderly people have been indicated as causes of poor denture hygiene14,20,22,26 . biofilm removal can be obtained by means of mechanical methods ( brushing and ultrasonic devices ) associated with chemical methods ( alkaline peroxide and hypochlorite , acids , enzymes and disinfectants ) ; among them , brushing is the most common method applied for routine denture biofilm control1,2,17,19,35 . knowledge on materials and methods for quantification of denture biofilm is an important factor to be considered . while biofilm quantification on natural teeth is significantly studied and published in the literature , its quantification on complete dentures is poorly known , due to the small number of papers published on this matter and because the procedure is not routinely employed by dentists16,24,27,28 . with respect to natural teeth , efficient hygiene control in complete dentures can be obtained by an orientation program , correct use of materials and methods available for denture cleansing and by utilization of a biofilm disclosing agent , allowing quantification and localization of biofilm on dentures , which could allow its removal more effectively5,8,12,16,24,26,28,31,35 . the aim of this study was to evaluate the efficacy of a biofilm disclosing agent associated with three brushes on hygiene control and maintenance in complete dentures . after research approval by the institutional review board of ribeiro preto dental school - university of so paulo ( no . 2000 - 136358.0 ) , 27 healthy men and women wearing complete denture ( maxillary and mandibular complete dentures ) were selected , with approximate mean ages of 50 years , and with no motor deficiency according to the discipline of complete denture of forp - usp . patients ' maxillary complete dentures were constructed with heat- cured acrylic resin , acrylic teeth , none of them with any break or repair , wearing time ranging from 1 to 3 years and score of biofilm degree of at least 1 ( additive index 2 ) . patients were included in the research after being verbally informed , when they read and signed the consent term . patients were divided into three groups : toothbrush ( oral b40 - service industry and commerce ltd . , so paulo , so paulo , brazil ) ; denture brush ( johnson & johnson - johnson & johnson , so paulo , brazil ) ; and denture brush ( denture - condor s.a . , santa catarina , brazil ) . for all groups , a specific denture paste ( dentu - creme , dentco , inc . two hygiene techniques were indicated for all groups ; technique i : the subjects were instructed to brush their dentures three times a day , rinsing their mouth with water after brushing and keeping the denture immersed in water overnight ; technique ii : the instruction was to brush the denture 3 times a day associated with the use of a disclosing agent ( 1% neutral red ) to the last brushing of the day ; in this technique , patients were also instructed to rinse their mouth with water after brushing and to keep the denture immersed in water overnight . after the patients had received instructions and hygiene materials , they were assessed by the investigators , who performed disclosure of the internal surface of maxillary dentures with 1% neutral red solution . the investigator brushed the disclosed dentures until complete removal of the disclosed biofilm ( biofilm - free ) . at first , technique i was employed by all patients for 3 weeks ; after this period , the investigator accomplished complete denture biofilm removal by brushing ( biofilm - free ) and then technique ii was employed by all patients for further 3 weeks . during each technique , the patients attended weekly returns when the maxillary denture was removed , rinsed with running water ( 5 seconds ) and air - dried ( 10 seconds ) . afterwards , the disclosing agent ( 1% neutral red ) was applied on the internal surface with a cotton swab and the denture was rinsed and dried again . the disclosed surfaces were photographed ( digital camera , coolpix , nikon , melville , n.y . , the camera was fixed on a stand ( cs-4 copy stand testrite , newark , nj , usa ) at 90 to the internal denture surface . after photographing , dentures were brushed with denture brush , in order to perform full biofilm removal , and then returned to the patients . biofilm quantification was performed at the end of each hygiene technique by a computerized method . by this method , the total internal surface of the denture and biofilm - covered areas were measured using the image tool software ( windows , version 2.02 , uthsc , san antonio ) . the percentages of biofilm coverage areas were calculated as the ratio between disclosed areas and the area of denture 's internal surface multiplied by 100 . three measurements were performed ( one for each visit ) of biofilm degree for each treatment ( i and ii ) . preliminary normality and homogeneity tests applied to the results showed no normal distribution ; therefore , the wilcoxon test was employed to compare the effectiveness of both techniques for each brush group ; the kruskal wallis test was employed to compare the effectiveness of brushes . these tests were applied to the average of biofilm percentage for each denture in each treatment ( 3 measurements ) . table 1 shows the mean of three measurements of biofilm percentage on the internal surface of the evaluated complete dentures and the sum of means . table 2 shows the statistical significance between techniques i and ii ( wilcoxon test ) . table 3 shows the results of kruskal wallis test applied to the means ( % ) shown in table i ; this test performs comparisons between brush groups with both techniques . ( ns ) : non - significant figure 1 shows the comparison between the sum of means obtained for three brushes in each technique , and compares the techniques with each other the relationship between denture biofilm , mucosal inflammation and atrophic chronic candidiasis has been discussed along the years ; there is confirmation that cleansing can help to control or solve the inflammatory conditions of the mucosa by reducing the degree of denture biofilm . nevertheless , several studies call attention to the precarious oral health of complete denture wearers1,5,11,16,17,19,23,34 . the toothbrush is the most important element in any hygiene program and in complete denture hygiene control ; brushing is the most common and routine method employed in association with paste as an auxiliary agent . some studies have shown that toothbrushes ( conventional brushes ) can cause wear on the denture materials and do not provide adequate hygiene , since their design is inappropriate to reach the entire denture surfaces ( internal and external)7,9,32,33 . such materials are customarily found in other countries , but in the brazilian market there is only a limited number of specific denture products ; as a result , denture wearers make use of conventional brushes to clean their dentures . studies regarding natural teeth hygiene emphasize the importance of incentive programs on the control of biofilm formation and many of these indicate home use of disclosing agents as additional means to brushing14,16,25 . in the reviews , complete denture studies indicate the use of these solutions only to quantitatively and qualitatively evaluate the denture biofilm2,3,4,12,17,22,24,28 as a mean to evaluate hygiene products . therefore , it is necessary compare the effectiveness of brushes that are available in the brazilian market , looking for their viable association with alternative methods , as well as using a disclosing agent to make biofilm removal easier and more effective . oral b toothbrush ( conventional ) has established good acceptance and regularity in the brazilian market along the years . it is composed of 41 soft bristles positioned perpendicularly at the same side of brush 's head , which has an oval design . the johnson & johnson brush was designed by paranhos , et al.30 ( 2000 ) to evaluate a specific dentifrice on the control of atrophic chronic candidiasis in denture wearers . it has 26 tufts with 16-mm long bristles positioned at the same side of the brush 's head . the longer length makes the bristles become extra - soft and provides a better range to reach the denture areas . the denture brush has a long handle , the head is elongated and has two groups of soft bristles positioned at each side of the head . one of them is more compact with a wedge - like form and is positioned on the extremity of the head . this bristle displays an anterior angulation and is designed to perform brushing on the internal surface of denture . the other group has a larger number of bristles , positioned perpendicular to the head covering its full extension and is designed to perform brushing on the denture 's external surface . with regard to the biofilm quantification method on complete dentures , disclosure is the most employed method in the literature and is customarily associated with the score attribution method ( index ) . nevertheless , some components of this methodology allow variations in results when utilized by different examiners . in this study , the biofilm quantification methodology was based on utilization of disclosed surfaces and a computerized method ( image toll 2.02 ) to quantify biofilm . the image tool 2.02 program was employed to obtain final results , since this method supplies numerical data that avoid percentage calculations of the biofilm coverage area . assessment was performed on the internal denture surface , since it is the area most often used to evaluate cleanser efficacy . lovato , et al.21 ( 2000 ) , studying clinical manipulation of a disclosing agent , highlighted the fact that this agent must have the ability to disclose biofilm , be easy to remove from the denture , and not stain the denture after removal . these authors indicated 1% neutral red , 1% sodium fluorescein , replak and 1% monosulphate proflavim as disclosing agent . the 60-day experimental period was divided between technique i ( brushing ) and technique ii ( brushing and disclose home application ) . before starting technique i , the denture was disclosed and the biofilm was fully removed by brushing ( denture - brush - condor ; dentu - crme , dentco ) by the investigator . at this moment , the biofilm was photographed , because it was not the purpose of this study to evaluate the improvement of denture hygiene related to the patients ' hygiene habits , but rather to evaluate the efficacy of techniques i and ii used in this study in association with different brushes . all 3 brush groups started the experiment with technique i , which was employed for 3 weeks . after the end of technique i , the patients were instructed to return to their habitual hygiene for 15 days so that the investigator could repeat disclosing and brushing of the maxillary complete dentures , to provide full biofilm removal ( biofilm - free ) before initiation of technique ii ( 3 weeks ) . this 15-day interval was important to reduce the influence from one technique on the other . throughout the entire experimental period , the patients returned weekly for biofilm quantification , since 7 days is an appropriate period for new biofilm deposition and to allow for good patient control . the 1% neutral red was selected due to its good affinity with biofilm , removal efficacy from denture surface and because it does not damage the denture components ( teeth and base ) . regarding the efficacy of brushes during technique ii , no significant difference was found among the three groups . these results indicate that it is not necessary to use a specific brush when an auxiliary agent for biofilm identification was employed prior to brushing . by using disclosing this can be explained by the fact that disclosure promoted biofilm visualization by the patient 5,18,20,25 . in agreement with kipot , et al.18 ( 1984 ) , it is essential that patients be instructed , trained and motivated to continue adequate oral hygiene . the increase in life expectancy of the brazilian population further increases the needs of edentate patients . this study aimed at contributing with maintenance of patients ' health , since there is direct association between denture biofilm control and associated pathologies as chronic atrophic candidiasis . home use of a disclosing agent improved biofilm control in dentures for all 3 groups of brushes . denture brush was more effective than the others in technique i , while there was no difference among brushes in technique ii . therefore , without the use of a disclosing biofilm agent , a specific brush for denture hygiene should be indicated . on the other hand , any type of brush can be used if associated with home use of a disclosing agent .

objective : this report evaluated the efficacy of three brushes and one biofilm disclosing agent in complete denture cleansing . methods : twenty - seven wearers of maxillary dentures were distributed into three groups and received different brushes : oral b40 , conventional toothbrush ( oral b ) ; denture , denture - specific brush ( condor ) ; johnson & johnson , denture - specific brush ( johnson & johnson ) . the 60-day experimental period was divided into two techniques : i - brushing ( brush associated with a paste - dentu creme , dentco ) three times a day ; ii - brushing and daily application of 1% neutral red on the denture internal surface . biofilm quantification was carried out weekly and the areas with dye biofilm were obtained by means of image tool 2.02 software.results:biofilm removal was more effective during technique ii ( wilcoxon test : p=0.01 ) for the three groups of brushes . when the brushes were compared in technique i , the kruskal wallis test indicated statistical difference between denture x johnson & johnson and denture x oral b40 , in which the denture was more efficient . for technique ii , there was no statistical difference between brushes ( p>0.05).conclusion : the disclosed application promoted more efficacy on biofilm removal , regardless of the brush used . denture ( condor ) was more efficient than the other brushes during technique i.

Objective: Results: Conclusion: INTRODUCTION MATERIAL AND METHOD RESULTS DISCUSSION CONCLUSION

PMC3557627

the world health organization ( who ) has defined osteoporosis as a skeletal disorder characterized by diminished bone strength resulting in increased fracture risk . bone strength is determined by interacting somatic and genetic factors . reported somatic factors include aging [ 25 ] , menopause [ 5 , 6 ] , and body mass index ( bmi ) [ 4 , 5 , 7 , 8 ] . to identify the genetic determinants of osteoporosis , an earlier study by the first author of this paper investigated how the incidence of low bone mineral density ( bmd ) in taiwanese women is affected by interactions among eleven single nucleotide polymorphisms ( snps ) in nine genes known to be involved in osteoporosis [ 1016 ] , including tumor necrosis factor - alpha ( tnf ) , transforming growth factor - beta 1 ( tgfb1 ; tgf1 ) , osteocalcin , parathyroid hormone ( pth ) , interleukin 1 receptor antagonist ( il1_ra ) , heat shock 70 kda protein 1-like ( hspa1l ; hsp70 hom ) , heat shock 70 kda protein 1b ( hspa1b ; hsp70 - 2 ) , calcitonin receptor ( ctr ) , and bone morphogenetic protein-4 ( bmp-4 ) . generally , several hormones , cytokines , and cell signaling - related proteins were chosen . for example , ctr , which is a receptor for the linear polypeptide hormone calcitonin , reduces blood calcium and suppresses the effects of pth . the cytokine family includes tnf , tgf- , bmp4 ( protein of tgf-superfamily ) , and il-1ra ( protein of interleukin 1cytokinefamily ) whereas cell - signaling proteins include hsp70 hom and hsp70 - 2 . studies of the interactions among these hormones ( e.g. , and references therein ) indicate that osteoporosis is an endocrinological problem . accumulating evidence reveals that snps are potential genetic markers for predicting osteoporosis outcome in taiwanese women . chang et al . also proposed a novel odds ratio - based genetic algorithm ( or - ga ) method of using odds ratios for quantitatively measuring the disease risk associated with various snp combinations to determine the susceptibility to osteoporosis in taiwanese women . taiwanese women who are carriers of risk alleles in two or more of these snps are likely to be at increased risk of osteoporosis because several partial deficiencies in these pathways may severely diminish bone density . therefore , snps may indicate risk of osteoporosis in taiwanese women and may be useful in clinical association studies to determine the genetic basis of disease susceptibility . the risk of osteoporosis is likely to be higher than normal in carriers of risk alleles in two or more of these snps because several partial deficiencies in these pathways may substantially decrease bone density . therefore , interacting polymorphisms may affect osteoporosis risk . in , the effects of age , bmi , and eleven interacting polymorphisms in nine genes were studied in terms of their effects on the incidence of low bmd ( table 1 ) . the findings showed that specific snp combinations may be risk factors for postmenopausal osteoporosis in taiwanese women . in addition to these specific snp combinations , bmi and age also showed independent associations with bmd in postmenopausal taiwanese women . although an apparent association between snps and osteoporosis has been identified in taiwanese women , a continuing challenge in genomics studies of taiwanese women populations lies in identifying significant genes . exhaustive computation over the model space is infeasible if the model space is very large , as there are 2 models with p snps [ 20 , 21 ] . feature selection techniques are designed to find responsible genes and snps for certain diseases . by selecting a small number of snps with significantly larger effects compared to other snps and by disregarding snps of lesser significance , researchers can focus on the most promising candidate genes and snps for use in diagnosis and therapy [ 21 , 22 ] . in , combined polymorphisms in different genomic regions the findings showed that a combination of several gene polymorphisms contributes to the development of osteoporosis in taiwanese women . however , that study did not report a subset of snps that can be used to predict osteoporosis outcome in this population . therefore , the current study used the same dataset used in to elucidate the relationship between osteoporosis and snps in taiwanese women in a performance comparison of three different classification algorithms with wrapper - based feature selection : multilayer feedforward neural network ( mfnn ) [ 2428 ] , naive bayes , and logistic regression . the mfnns have proven particularly effective for nonlinear mapping based on human knowledge and are now attracting interest for use in solving complex classification problems . an mfnn containing layers of simple computing nodes , which is analogous to brain neural networks , has proven effective for approximating nonlinear continuous functions and for revealing previously unknown relationships between given input and output variables [ 25 , 26 ] . the unique structure of mfnns enables them to learn by using algorithms such as backpropagation and evolutionary algorithms [ 31 , 32 ] . potential medical applications of mfnns include solving problems in which the relationship between independent variables and clinical outcome are poorly understood . because mfnns are capable of self - training with minimal human intervention , many studies of large epidemiology databases have , in addition to conventional statistical methods , used mfnns for further insight into the interrelationships among variables . a naive bayes classifier assumes that the presence ( or absence ) of a particular feature of a class is unrelated to the presence ( or absence ) of any other feature , given the class variable . depending on the precise nature of the probability model , naive bayes classifiers can be trained very efficiently in a supervised learning setting . the classifier obtained by using this set of discriminant functions and by estimating the relevant probabilities from the training set is often called the naive bayesian classifier because , if the the attributes are naively assumed to be independent given the class , direct application of the bayes theorem easily confirms that this classifier is optimal in terms of minimizing the misclassification rate or zero - one loss [ 34 , 35 ] . logistic regression is a statistical method of predicting the outcome of a variable that is categorical ( i.e. , it can have several different categories ) and is dependent on one or more predictor variables . a logistic function can be used to model the probabilities describing the possible outcome of a single trial as a function of explanatory variables . logistic regression is typically used to measure the relationship between a categorical dependent variable and one or more continuous independent variables by converting the dependent variable to probability scores . the wrapper - based feature selection method , in which the feature selection algorithm acts as a wrapper around the classification algorithm , was also used to identify an snp subset with sufficient predictive power to distinguish between high- and low - risk alleles . in the wrapper - based approach , the function used to evaluate feature subsets uses the classification algorithm itself to perform a best - first search for a good subset . starting from an empty feature set , it searches forward for potential feature subsets by performing greedy hillclimbing augmented with a backtracking technique . . showed that it may be superior to hybrid approaches combining chi - square and information - gain methods reported in the literature . a comprehensive literature review shows no attempts to predict osteoporosis outcome in taiwanese women using genetic factors ( snps ) and the three above mentioned classification algorithms with wrapper - based feature selection method . this study therefore compared performance in three classification algorithms : mfnn , naive bayes , and logistic regression , with and without wrapper - based feature selection techniques . identifying the genes and snps associated with taiwan population of women with osteoporosis would enable researchers to focus on the candidate genes and snps that are most promising for use in diagnosis and therapy . the results of our studies could be generalized to snp searches in genetic studies of human disorders and to development of new molecular diagnostic / prognostic tools . however , before routine application of genomic analysis in clinical practice , genetic markers must be validated in prospective clinical trials . the dataset in this study , which included snps , age , menopause , and bmi , was the same dataset used in a previous study by the first author of this paper . the t - score was calculated according to who classifications using a locally derived reference range provided by the manufacturer . the subjects were divided into two bmd groups according to t - score [ 3840 ] . subjects with t - score > 1 were enrolled in the high bmd group , and those with t - scores 1 were enrolled in the low bmd group . the overall dataset was derived from 295 cases , including ( i ) 247 postmenopausal cases ( 83.73% ) and 48 prepremenopausal cases ( 16.27% ) ; ( ii ) 112 high bmd cases ( 37.97% ) and 183 low bmd cases ( 62.03% ) . post - menopause was defined as the absence of menstruation for > 6 months or age 50 years . clinical data used for diagnosis were further converted into numerical form , that is , 1 for high bmd and 0 for low bmd . table 1 shows the 22 snps analyzed in this study , which were the same as those analyzed previously by the first author of this paper . table 1 shows that the nine candidate genes included tnf , transforming growth factor - beta 1 ( tgf1 ) , osteocalcin , parathyroid hormone ( pth ) , interleukin 1 receptor antagonist ( il1_ra ) , hsp , calcitonin receptor ( ctr ) , bone morphogenetic protein-4 ( bmp-4 ) , and three genotypes per locus . the three families of classification algorithms used as the basis for comparisons in this study were mfnn , naive bayes , and logistic regression . these classifiers were implemented using the waikato environment for knowledge analysis ( weka ) software . an mfnn is an artificial neural network ( ann ) model in which connections between the units do not form a directed cycle [ 2428 , 30 ] . from an algorithmic perspective , the underlying process of an mfnn can be divided into retrieving and learning phases . assume an l - layer feedforward neural network with nl units at the lth layer . in the retrieving phase , the mfnn iterates through all layers to produce the retrieval response { ai(l ) , i = 1 , 2 , , nl } at the output layer based on test pattern inputs { ai(0 ) , i = 1 , 2 , , n0 } , the known weights wij of the network , and the nonlinear activation function fi ( e.g. , sigmoid function ) . in the learning phase of this mfnn , the backpropagation algorithm and evolutionary algorithms [ 31 , 32 ] are used in the learning scheme . the weight updating mechanism is a backpropagation of corrective signals from the output layer to the hidden layers . the goal is iteratively selecting a set of weights wij(l ) for all layers such that the squared error function e can be minimized by a pair of input training patterns { ai(0 ) , i = 1 , 2 , , n0 } and target training patterns { tj , j = 1 , 2 , , nl}. mathematically , the iterative gradient descent formulation for updating each specific weight wij(l ) can be expressed by the following equation : ( 1)wij(l)wij(l)ewij(l ) , where is the learning rate and e/wij(l ) can be effectively calculated through a numerical chain rule by backpropagating the error signal from the output layer to the input layer . structurally , however , an mfnn is a spatial and iterative neural network with several layers of hidden neuron units between the input and output neuron layers . the basic function of each neuron is the linear basis function , and activation is modeled with a non - decreasing and differentiable sigmoid function . inputs contain the information about clinical factors , for example , snps , that are needed for the database . the mfnn is trained first by repeatedly providing input - output training pairs and by executing the backpropagation learning algorithm . after this training process is complete , the mfnn is tested by sending testing data inputs ( i.e. , snps ) to the network . the forward propagation of the mfnn reveals the osteoporosis outcome for a specific case so that causes can be inferred from effects . here , the default weka parameters were used , that is , hidden layer neurons = 6 , learning rate = 0.3 , momentum variable = 0.2 , and training time = 500 . second , all features in naive bayes , which is the simplest bayesian network , are assumed to be conditionally independent . let ( x1 , x2 , , xp ) be features ( i.e. , snps ) used to predict class c ( i.e. , disease status , 1 = high bmd or 0 = low bmd ) . given a data instance with genotype ( x1 , x2 , , xp ) , the best prediction of the disease class is given by class c , which maximizes the conditional probability pr(c = c | x1 = x1 , x2 = x2 , , xp = xp ) . bayes theorem is used to estimate the conditional probability pr(c = c | x1 = x1 , x2 = x2 , , xp = xp ) , which is decomposed into a product of conditional probabilities . third , the logistic regression generates the coefficients for the following formula used for logit transformation of the probability of a patient having a characteristic of interest : logit(p ) = b0 + b1x1 + b2x2 + +bkxk . the formula used to calculate the probability of the characteristic of interest in this study is p = 1/(1 + e ) , where 1 = high bmd and 0 = low bmd . the wrapper - based feature selection approach , in which a feature selection algorithm acts as a wrapper around a classification algorithm , was used to find a subset of snps that maximizes the performance of the prediction model . figure 1 shows that , in the wrapper approach , the feature subset is selected by using a black box classification algorithm ( i.e. , selection is performed using the interface alone and does not require knowledge of the algorithm ) . to search for a good subset , the search space is organized such that each state represents a feature subset . for n features , each state has n bits , and each bit indicates whether a feature is present ( 1 ) or absent ( 0 ) . to determine the connectivity between the states , this study used operators that add or delete a single feature from each state , where the states correspond to the search space commonly used in stepwise method . figure 2 shows an example of the state space and operators obtained by stepwise method in a four - feature problem . the classification algorithms are used to calculate a performance measure for each of 16 different subsets . therefore , the wrapper - based approach conducts a best - first search for a good subset by including the classification algorithm itself ( mfnn , naive bayes , or logistic regression ) in the feature subset evaluation . to search for potential feature subsets , the best - first search starts from an empty feature set and searches forward by greedy hillclimbing augmented with a backtracking technique . figure 3 shows how mfnn , naive bayes , and logistic regression were applied in the wrapper - based approach . the performance of the prediction models was measured in terms of receiver operating characteristic ( roc ) and area under the roc curve ( auc ) . the auc of a classifier can be interpreted as the probability of the classifier ranking a randomly chosen positive example higher than a randomly chosen negative one . most researchers have now adopted auc for evaluating the predictive capability of classifiers since auc is a better performance metric compared to accuracy . this study used the auc value for performance comparison of different prediction models using the same dataset . the higher the auc , the better the learning performance . other calculations included sensitivity , the proportion of correctly predicted responders out of all tested responders , and specificity , the proportion of correctly predicted nonresponders out of all tested nonresponders . to investigate the generalization of the prediction models produced by the above algorithms , the repeated 10-fold cross - validation method was used the model was then trained with nine - tenths of the data and tested by the remaining tenth of data to estimate its predictive performance . each time , a different tenth of the data was used as testing data , and a different nine - tenths of the data were used as training data . finally , the average estimate over all runs was reported by running the above regular 10-fold cross - validation 100 times with different splits of data . in repeated 10-fold cross - validation testing , the performance of all models was evaluated with and without feature selection . tables 3 and 4 summarize the results of the repeated 10-fold cross - validation experiments for mfnn , naive bayes , and logistic regression using snps with and without feature selection . first , the auc , sensitivity , and specificity were calculated for the three predictive models without wrapper - based feature selection . table 3 shows that the average auc values for the mfnn , the naive bayes , and the logistic regression prediction models were 0.489 , 0.462 and 0.485 , respectively . in terms of auc , the the mfnn model ( auc = 0.489 ) outperformed the naive bayes ( auc = 0.462 ) and logistic regression ( auc = 0.485 ) models . a repeated 10-fold cross - validation experiment was performed to compare performance in the three wrapper - based predictive algorithms . table 4 shows that the mfnn , the naive bayes , and the logistic regression models had average auc values of 0.631 , 0.569 , and 0.620 , respectively . in terms of auc , the mfnn model ( auc = 0.631 ) outperformed both the naive bayes model ( auc = 0.569 ) and the logistic regression model ( auc = 0.620 ) . out of 11 snps , the wrapper - based mfnn model identified only 4 : rs1800469 ( tgf1 - 509 ) , vntr ( il1_ra ) , rs2227956 ( hsp70 hom ) , and rs1801197 ( ctr ) . feature selection using the wrapper - based approach clearly improved performance in the mfnn , the naive bayes , and the logistic regression . overall , the mfnn classifier with the wrapper - based approach demonstrated superior prediction performance ( auc = 0.631 ) compared to the other models . additionally , the mfnn classifier with wrapper - based feature selection required fewer snps ( n = 4 ) compared to the mfnn classifier without feature selection ( n = 11 ) . table 4 shows that the aucs did not significantly differ between the mfnn model with wrapper - based feature selection ( auc = 0.631 ) and the logistic regression model with wrapper - based feature selection ( auc = 0.620 ) . however , the mfnn classifier with wrapper - based feature selection required fewer snps ( n = 4 ) compared to the logistic regression classifier with wrapper - based feature selection ( n = 8) , that is , by selecting a small number of snps with significantly larger effects compared to other snps and by disregarding relatively insignificant snps , the mfnn model with wrapper - based feature selection successfully identified a subset of four major snps that could be used to predict osteoporosis outcome in the study population ( rs1800469 ( tgf1 - 509 ) , vntr ( il1_ra ) , rs2227956 ( hsp70 hom ) , and rs1801197 ( ctr ) ) . after confirming that the mfnn model outperforms the logistic regression model , the next objective was finding the candidate genes and snps that are most promising for diagnosing osteoporosis , designing therapies , and predicting outcome in the studied population of taiwanese women with osteoporosis . this study compared three classification algorithms , including mfnn , naive bayes , and logistic regression with and without feature selection in terms of accuracy in predicting osteoporosis outcome in a population of taiwanese women . accounting for models is not a trivial task because even a relatively small set of candidate genes obtains a large number of possible models . the three classifiers were chosen for comparison because they cover varying techniques with different representational models such as probabilistic mfnn , naive bayes , and logistic regression models . the proposed procedures can also be implemented using the publicly available software weka and are thus easily applicable in genomic studies . to the best of our knowledge , this study is the first to propose the use of three classification algorithms , including mfnn , naive bayes , and logistic regression , and wrapper - based feature selection method for modeling osteoporosis outcome in taiwanese women based on genetic factors such as snps . in this paper , the wrapper - based feature selection approach was used to find a subset of snps that maximizes the performance of the prediction model according to how feature selection search is incorporated in the classification algorithms . the results showed that the mfnn classifier with wrapper - based approach was superior to the other tested algorithms and achieved the greatest auc with the smallest number of snps when distinguishing between high and low bmd in taiwanese women . these results suggest that mfnn model is a good method of modeling complex nonlinear relationships among clinical factors and the responsiveness of osteoporosis outcome in taiwanese women . the wrapper - based approach does not require knowledge of the classification algorithm used in the feature selection process , in which features are optimized by using the classification algorithm as part of the evaluation function [ 21 , 23 ] . another advantage of the wrapper - based method is its inclusion of the interaction between feature subset search and the classification model . however , the risk of over - fitting is high when using the wrapper - based method [ 21 , 45 ] . in the current study , use of the wrapper - based feature selection approach to assess high and low bmd individuals revealed a panel of genetic markers , including tgf1 - 509 , il1_ra , hsp70 hom , and ctr , which were more prominent compared to other markers observed in the examined taiwanese women population with osteoporosis . a noted limitation of this study is that , due to the small sample size , the auc values were too low ( < 0.7 ) to obtain good dataset classifications . therefore , further prospective clinical trials are recommended to determine whether the observed outcome associations with these candidate genes are reproducible in a larger population of taiwanese women with osteoporosis . this study used an mfnn methodology with wrapper - based feature selection method to predict osteoporosis outcome in taiwanese women based on clinical factors such as snps . the findings suggest that patients and doctors can use the proposed tool to enhance decision making based on clinical factors such as snp genotyping data . however , genetic markers require validation in further prospective clinical trials before routine clinical use of genomic analysis for predicting osteoporosis outcome .

an essential task in a genomic analysis of a human disease is limiting the number of strongly associated genes when studying susceptibility to the disease . the goal of this study was to compare computational tools with and without feature selection for predicting osteoporosis outcome in taiwanese women based on genetic factors such as single nucleotide polymorphisms ( snps ) . to elucidate relationships between osteoporosis and snps in this population , three classification algorithms were applied : multilayer feedforward neural network ( mfnn ) , naive bayes , and logistic regression . a wrapper - based feature selection method was also used to identify a subset of major snps . experimental results showed that the mfnn model with the wrapper - based approach was the best predictive model for inferring disease susceptibility based on the complex relationship between osteoporosis and snps in taiwanese women . the findings suggest that patients and doctors can use the proposed tool to enhance decision making based on clinical factors such as snp genotyping data .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

PMC2902048

interval mapping and multiple interval mapping are the most commonly used methods for qtl mapping . these methods are developed in the maximum likelihood framework , which has limitation in terms of handling large saturated models . bayesian mapping [ 37 ] deals with large models more efficiently through the reversible jump markov chain monte carlo ( rjmcmc ) , the shrinkage analysis [ 8 , 9 ] , or the stochastic search variable selection ( ssvs ) . shrinkage mapping and ssvs are more efficient in terms of whole genome evaluation because they are statistically easy to understand and also provide better chance to evaluate the entire genome . these two methods are related to the lasso method for regression analysis . rather than deleting nonsignificant qtl explicitly from the model , these methods use a special algorithm to shrink estimated qtl effects to zero or close to zero . a qtl with zero estimated effect is treated the same as being excluded from the model . no statistical test is required because genome regions bearing no qtl often show no bumps ( qtl effects ) in the qtl effect profile ( plot of qtl effects against genome location ) . the visual inspection on the qtl effect profile is not optimal because small qtl may come and go during the mcmc sampling process . it is desirable to provide some kind of statistical confidence on these small qtl . permutation test itself is not a method of qtl mapping ; rather , it is a method to find the critical value used to declare the significance of qtl for any method of qtl mapping . a new resampling method was developed by zou et al . for significance test under the composite interval mapping or other multiple effect based qtl mapping schemes . the new resampling method is computationally less intensive and may perform better than the permutation test . the reason for this is perhaps due to the fact that the theory behind the method is not straightforward to most qtl mapping experimentalists . the permutation test , people tend to trust a simple method they understand , rather than a comprehensive method they do not , even if the simple method is suboptimal . therefore , the permutation test remains the most popular method for finding the critical value of a test statistic for qtl detection . kopp et al . applied the permutation test to determine empirical thresholds for bayesian shrinkage mapping . the problem with such a test for the mcmc implemented bayesian mapping is the heavy computational burden . each mcmc run may take one or a few hours to complete for a reasonable sample size of the mapping population . performing thousands of permutation analyses broman and speed treated multiple qtl mapping as a model ( variable ) selection problem and developed a new method called bic. more recently , manichaikul et al . extended the broman and speed model selection by allowing epistatic ( nonallelic interaction ) effects to be included in the model . they called the extended model selection method the penalized lod score method ( plod ) . two versions of the penalized lod score method were investigated ; one is called the heavy penalized lod score ( plodh ) and the other is called the light penalized lod score ( plodl ) . with this new notation , the original bic of broman and speed was renamed as ploda , penalized lod score for additive effects only . the authors compared these methods along with two other bic - based methods and the bayesian model selection method of yi et al . using both simulated data and real data . they concluded that the plod methods including epistatic effects and the bayesian model selection method outperformed other methods in most cases they evaluated . they can not replace the bayesian shrinkage analysis because the two have quite different purposes . we realized that if the bayesian shrinkage analysis is accompanied with a significance test , it can serve both qtl detection and genome selection . the original bayesian shrinkage analysis [ 8 , 9 ] has no significance test associated with the method because the entire genome was evaluated simultaneously in a single model . more recently , researchers , especially animal and plant breeders , became interested in genome selection [ 18 , 19 ] using the bayesian method . genome selection does not require statistical tests because qtl of the entire genome , regardless the sizes , are included to predict the genomic effect of individuals . however , there is no report so far to investigate whether inclusion of small qtl will benefit genome selection . cross - validation can be used to determine how large a qtl should be included in genome selection . for the paper to be self contained , we briefly introduce the bayesian shrinkage model here . let yj be the phenotypic value of a quantitative trait measured from individual j for j = 1 , suppose that the individual is genotyped for m markers , which are more or less evenly distributed across the genome . let xjk be the genotype indicator variable for individual j at marker k for k = 1 , , m. the linear model describing the relationship between the phenotype and the genotypes of markers is ( 1)yj = b0+k=1mxjkbk+ej , where b0 is the intercept , bk is the qtl effect for marker k , and ej is the residual error with an assumed n(0 , ) distribution . the reason that the bayesian shrinkage method can handle a large m is the prior distribution assigned to each qtl effect : ( 2)p(bk)=n(bk 0,k2 ) , where k is a qtl specific prior variance . this prior alone is not sufficient to generate the desired shrinkage estimate of qtl effect . a hierarchical model with a higher level of prior assignment is necessary , in which the prior variance k is further assigned a scaled inverse chi - square distribution : ( 3)p(k2)=inv2(k2 , ) . in the original shrinkage analysis , xu set = = 0 , leading to p(k ) = 1/k . ter braak et al . claimed that this prior is improper and leads to an improper posterior distribution . they revised the prior so that the posterior distribution becomes proper . their revised prior is ( 4)p(k2)=inv2(k2 2,0)1(k2)1 , where 0 < 0.5 . if = 0 , this revised prior would be equivalent to xu 's vague prior . however , xu 's vague prior is just excluded from the revised prior . in this study , as a precaution to avoid any potential problems caused by the improper posterior distribution of k . in the mcmc - implemented bayesian shrinkage analysis , we could have plotted a test statistic , say a t - test or an f - test , against the genome location . the empirical posterior standard deviation highly depends on the thinning rate of the markov chain and thus is always underestimated due to possible autocorrelation . therefore , we prefer to use the qtl effect profile rather than a test statistic profile . to determine the threshold values for the qtl effects under the null model , we employed a permutation test just like frequentists do in interval mapping . let y = { yj } be the vector of the phenotypic values ordered according to the individuals ' natural identification numbers , that is , the original dataset where the individuals ' phenotypes match their marker genotypes in the files . let y * = { yj * } be a randomly rearranged vector of phenotypes , called a permutation , in which the phenotypes do not match the marker genotypes . performing a bayesian shrinkage analysis on the permuted data by running a markov chain with a desired length , we obtain a posterior sample for all the parameters . for the parameters of interest , say the qtl effects , we record their values and save them in a file as one observation from one permutation analysis . the permutation analysis is repeated independently for a thousand times ; we then obtain a thousand observations for each of the interested parameters ( qtl effects ) . this sample contains observations from the empirical distribution of the null model ( no qtl effects ) . the 0.5 100% and ( 1 0.5 ) 100% percentiles of a parameter in the thousand permuted samples are the empirical critical values used to declare statistical significance for a qtl in the analysis of the original dataset ( phenotypes match the genotypes ) . each mcmc run may take an hour or so and a complete permutation experiment consisting of 1000 permutation analyses may take a month computing time . therefore , we will invent a more efficient permutation method to replace this traditional method of permutation . as the name of the method implies , this permutation strategy permutes the phenotypes in every hth iteration within a markov chain , where 1 h l and l is the length of the markov chain . if h = l , this approach is equivalent to the permutation - between - chains approach . the approach is implemented as follows . for each iteration , after all parameters are sampled , the phenotypes are reshuffled before the next round of sampling starts . the total length of the chain is not necessarily longer than a regular markov chain for the unshuffled data . therefore , a complete data analysis requires only two chains , one for the original data and one for the reshuffled data . the reshuffled chain provides the 0.5 100% and ( 1 0.5 ) 100% percentiles used as critical values of the qtl effects . the within - chain permutation is a strategy to generate the posterior distributions of the regression coefficients under the null model . if the genotypes do not match the phenotypes , the bayesian estimates ( posterior means ) of the regression coefficients are expected to be zero across all loci . the posterior variances are determined by the residual variance and the variance of the genotypic indicator variables , which are preserved in the permuted sample , regardless how frequent the phenotypes are reshuffled . as long as we can control the type i error for the entire genome and produce reasonable powers for all the large qtl , the permutation test should be admissible . genome selection aims to evaluating the genetic effect for the entire genome using dense markers for each individual . when all individuals in a population are evaluated , the genomic effects of different individuals can be compared and the best individuals are selected for breeding . how to combine the qtl mapping result with genome selection is an important but not yet answered question . we adopted a five - fold cross - validation test to answer this question . in the cross - validation analysis , we partition the sample into five equal parts ( subsamples ) . each time , we use four parts ( 4n/5 individuals ) to estimate the qtl effects and perform within - chain random shuffling to determine the empirical percentiles for qtl detection . only significant qtl at the level is used to predict the total genomic effect for an individual in the remaining part ( n/5 individuals ) . note that the training sample ( 4n/5 individuals ) is used for parameter estimation and significance test and the testing sample ( n/5 individuals ) is used for prediction . the squared prediction error ( pe ) for the s - part is defined as ( 5)s()=5nj=1n/5(yjb^0k=1mxkjb^k)2 , where yj is the phenotypic value of an individual in the test sample and j indexes all individuals in the test sample . note that b^k equals the shrinkage estimate if it passes the thresholds and b^k=0 otherwise . the overall pe for the cross - validation test is ( 6)pe()=15s=15s( ) . the value that minimizes the pe is the optimal one used as the criterion of qtl inclusion for genome selection . the design of the simulation experiment conducted by wang et al . was adopted here exepct that the population simulated was an f2 rather than a bc population . the sample size was fixed at 500 , which is a typical sample size used in most qtl mapping experiments . the genome size was 2400 cm long covered by 241 evenly distributed markers ( 10 cm per marker interval ) . a total of 20 qtls were placed on the genome and the positions and effects of the 20 qtl are presented in table 1 . the proportions of qtl explaining the total phenotypic variance were calculated based on the following method . the genotype indicator variable for individual j at locus k is defined as xjk = { 1 , 0 , 1 } for the three genotypes dominance effects were not simulated and also not included in the model for this simulation experiment because they do not help answer questions addressed in this study . these parameter values were used to generate a quantitative trait with a population mean b0 = 10.0 and a residual error variance = 10.0 . the total genetic variance for the trait is ( 7)vg=k=120 k=120bkbkcov ( zk , zk)=12k=120 k=120bkbk(12rkk ) , where rkk is the recombination frequency between qtl k and k , cov ( zk , zk ) = var(z)(1 2rkk ) is the covariance between zk and zk , and var(z ) = 1/2 is the variance of z ( assuming no segregation distortion ) . the total genetic variance for the quantitative trait is vg = vq + vl = 66.384 , which is the sum of the genetic variances due to qtl ( vq ) and covariance between linked qtl ( vl ) , where ( 8)vq=12k=120bk2=46.7804,vl=k>k20bkbk(12rkk)=19.6034 . the proportion of the genetic variance contributed by each qtl is 0.5bk / vg for the kth qtl ( given in the column headed with prop - g in table 1 ) . the corresponding proportion of the phenotypic variance contributed by the kth qtl is 0.5bk / vp and given in the column headed with prop - p in table 1 . all 241 markers were included in the model , leading to the dimensionality of the model of n ( m + 1 ) = 500 ( 241 + 1 ) . the burn in period was 1000 . the chain was thinned by keeping one observation out of 10 iterations until the posterior sample size reached 5000 . the true values of the qtl effects and the locations of the simulated qtl are depicted in table 1 . say qtl numbers 19 and 20 , the estimated effects are also small with values no larger than the bumps in the no qtl regions ( noises ) . we calculated the equal tail credible interval at = 0.05 , that is , the 2.5%97.5% percentile range , for each marker . only one ( the largest ) qtl was detected because the interval excluded 0 ( data not shown ) . the equal - tail credible intervals of all other qtl covered zero , and thus , they are not significant in terms of statistical testing . using the equal tail credible interval at = 0.10 , two more qtls were detected in addition to the largest qtl ( data not shown ) . certainly , the equal tail credible interval is not a good criterion for significance test . the posterior distributions for most estimated qtl effects have a special distribution with a spike at zero , which is the cause for the failure of equal tail credible interval as the criterion for significance test . the reason is that almost all qtls have an equal - tail interval covering the null value , for example , zero . even the largest qtl in our simulation had a high probability mass at zero ( see figure 2 ) . this spike - shaped or zero - inflated posterior distribution for qtl effect is typical in bayesian shrinkage mapping . if we had used the equal tail interval at = 0.05 as the significance test criterion , only one qtl ( the largest one ) , out of the 20 simulated qtl , would have reached the statistical significance level . the permutation test , however , detected many major qtl , as demonstrated next in the permutation test sections . each permuted sample was subject to the same mcmc analysis as the original data ( 51000 iterations ) . the sas / iml program took approximately 20 days in a dell pc ( 2.5 ghz and 3.25 go of ram ) . for each marker , the 2.5%97.5% and 5%95% intervals ( corresponding to = 0.05 and = 0.10 ) were calculated . the profiles of these percentiles along with the estimated qtl effects are given in figure 3(a ) . using the 2.5%97.5% interval , we can detect 15 qtl out of the 20 simulated qtl . a few more qtls with small effects the results here are more reasonable than those when the equal tail credible interval was used . this permutation strategy only requires running one more chain in addition to the mcmc run of the original data . we first evaluated the performance of h = 1 , that is , reshuffling the phenotype in every iteration . the 2.5% , 5% , 95% , and 97.5% percentiles plotted against the genome location are shown in figure 3(b ) to compare with the result of permutation outside the chains . these intervals ( the within - chain permutation ) appear to be wider than the intervals of the between - chain permutation analysis . therefore , the tests for the within - chain permutation are more conservative than the between - chain permutation . using the within - chain permutation , 13 qtls were detected for = 0.05 and 19 qtls were detected for = 0.10 , not too much different from the result of the between - chain permutation . a more conservative test is better than a more liberal test , as long as the statistical power is not compromised ( examined later in the power study section ) . the 2.5% , 5% , 95% , and 97.5% percentiles plotted against the genome location are shown in figure 4 . these intervals appear to be similar to h = 1 except that the higher h 's tend to generate rougher percentile profiles . therefore , h = 1 is more preferable than other values of h. hereafter , we chose h = 1 for all subsequent analysis . using the same parameters given in table 1 , we simulated 100 more independent samples to investigate the statistical power of the bayesian shrinkage method . one run was the mcmc sampler on the original data to estimate qtl effects and the other run was the mcmc sampler on the within - chain reshuffled data to generate the critical values for qtl detection . the statistical power for each qtl was calculated based on the proportions of samples in which the qtl fell outside the empirical intervals . we observed that if a true qtl failed to be detected at the locus where it was placed , the effect was often picked up by a marker nearby ( 10 cm away ) . therefore , a true qtl was claimed to be detected if one or more of the triplets ( three loci ) covering the true qtl ( 20 cm range ) was detected . the powers seem to be reasonable ; seven out of the 20 simulated qtl have a power reached 80% at = 0.10 . therefore , the conservative within - chain permutation significance test does not sacrifice much statistical power . for the 241 marker effects included in the model , were reserved for the true qtl ( 20 true qtl plus 40 flanking markers ) , leaving 241 60 = 181 model effects as false qtl . if a false qtl was detected in a particular sample , it was counted as one false positive . for each false qtl the proportion of false positive ( false positive rate or type i error ) was recorded for each false qtl simulated . the observed false positive rate is indeed less than 0.05 , confirming our previous conclusion that the within - chain permutation approach is conservative . the average false positive rate for all these markers was about 0.05 , again confirming the conservativeness of the within chain permutation approach . using the original data simulated in the beginning of the experiment ( not a sample from the power study ) , we performed the fivefold cross - validation study to determine how large a qtl should be included in the model to predict the total genetic value of an individual . the pe ( squared prediction error ) values are plotted against the value in figure 7 . the decrease of the pe from = 0.0 to = 0.2 is very sharp , but after = 0.2 , the pe value tends to be stabilized or slightly increased . the conclusion is that in genome selection , we should choose the value around 0.2 . of course we recommend such a cross - validation test for each data analysis to determine how many qtl should be included . from the pe profile , including all qtls ( = 1 ) into the prediction model ( regardless the sizes of the qtl ) does not lead to any significant loss in the precision of genome selection compared to the optimal number of qtl determined by the cross - validation test . therefore , a robust choice is to include all qtls in the model for genome selection . we now use three sample data to demonstrate the application of the permutation test - associated bayesian shrinkage analysis . these data were collected from qtl mapping experiments in model plants and agricultural crops . the first dataset is the recombinant inbred line data of arabidopsis data , where the two parents initiating the line cross were bay-0 and shahdara with bay-0 as the female parent . the recombinant inbred lines were actually f7 progeny of single seed descendants of the f2 plants . flowering time was recorded for each line in two environments : long day ( 16-hour photoperiod ) and short day ( 8-hour photoperiod ) . a couple of lines did not have the phenotypic records and their phenotypic values were replaced by the population mean for convenience of data analysis . these markers are more or less evenly distributed along five chromosomes with an average 10.8 centimorgan ( cm ) per marker interval . we inserted a pseudomarker in every 2 cm of the genome . including the inserted pseudomarkers , the total number of loci subject to analysis was 200 ( 38 true markers plus 162 pseudomarkers ) . therefore , the model for the short day flowering time trait is ( 9)y = b0+k=1200xkbk+ , where xk is a 420 1 vector coded as 1 for one genotype and 0 for the other genotype for locus k. if locus k is a pseudomarker , xk = pr(genotype = 1 ) , which is the conditional probabilities of marker k being of genotype 1 . finally , bk is the qtl effect of locus k. for the original data analysis , the burn - in period was 1000 . the posterior sample size was 10000 , and thus the total number of iterations was 1000 + 10000 10 = 101000 . the posterior sample size of the within - chain permutation analysis was 80000 , that is , 1000 + 80000 10 = 801000 iterations in total . the estimated qtl effects and the permutation generated 2.5%97.5% and 5%95% intervals are plotted in figure 8(a ) . chromosomes 1 and 4 each has one qtl and chromosome 5 has two qtl . when = 0.10 was used , one more qtl on chromosome 1 was detected . the fivefold cross - validation shows that the optimal strategy of genome selection for this dataset was to include all qtls in the model , regardless the significance of the estimated qtl effects ( see figure 8(b ) ) . the general pattern of the pe profile remains the same as that of the simulated data . below = 0.2 the decrease of pe was dramatic but after = 0.2 the pe values approached a stable value . the second data are the doubled haploid ( dh ) data obtained from luo et al . . this dataset consists of 150 doubled haploids ( dhs ) derived from the cross of two spring barley varieties , steptoe and morex , designated as the s m cross . the phenotype was the spot blotch ( a fungus cochliobolus sativus ) resistance measured as the lesion size on the leaves of barley seedlings . the total number of markers was 495 distributed along seven chromosomes of the barley genome . because of the small sample size , we could not analyze all the 495 markers simultaneously ( high collinearity ) . therefore , we placed one pseudomarker in every 5 cm and overall obtained 225 pseudomarkers for the entire genome . therefore , the model for the disease resistance trait is ( 10)y = b0+k=1225xkbk+ , where xk is a 150 1 vector coded as 1 for one genotype and 0 for the other genotype for locus k. if locus k is a pseudomarker , xk = pr(genotype = 1 ) , which is the conditional probabilities of marker k being of genotype 1 . finally , bk is the qtl effect of locus k. the parameters of the mcmc experiment ( e.g. , burn - in period , thinning rate , etc . ) the estimated qtl effects and the permutation - generated 2.5%97.5% and 5%95% intervals are plotted in figure 9(a ) . these two are major qtl because their estimated values are way over the critical value . when the critical values at a = 0.10 were used , five more qtls were declared as significant . the cross - validation analysis shows that the optimal strategy of genome selection for this dataset was to include all qtls that are significant at = 0.15 ( see figure 9(b ) ) . below = 0.15 the decrease of pe was dramatic but after = 0.15 the pe values increased slightly until they reached a plateau at = 0.3 ( see figure 9(b ) ) . this example demonstrated the usefulness of using cross - validation to select qtl for inclusion for prediction of genomic effect . this example demonstrates the application of the bayesian shrinkage analysis to qtl mapping for the number of seeded spikelets ( a female fertility trait ) in wheat . the experiment was conducted by dou et al . who made the data available to us for this analysis . a female sterile line xnd126 and an elite cultivar gaocheng 8901 with normal fertility were crossed for genetic analysis of female sterility measured as a quantitative trait . the f1 and f2 progeny of the parents were planted at the huaian experimental station in china for the 2006 - 2007 growing season under the normal autumn sowing condition . these markers covered 5 chromosomes of the wheat genome with an average genome marker density of 15.5 cm per marker interval . these chromosomes were scanned for qtl of the fertility trait using the mcmc implemented bayesian method . the dependent variable was the fertility phenotype while the independent variables were numerically coded genotype indicator variables for the part of genome under investigation . we placed one pseudomarker in every 5 centimorgan ( cm ) of the genome . therefore , we have a total of 75 independent variables . for each independent variable , the numerically coded value was the difference between the conditional probabilities of the two homozygote genotypes . let a1a1 , a1a2 , and a2a2 be the three genotypes for the kth pseudo marker of the genome . the numerically coded value for the locus is ( 11)xjk = p(gjk = a1a1 marker)p(gjk = a2a2 marker ) for k = 1 , , 75 . the map of the 75 pseudomarkers , the phenotypic values of the 243 plants , and the 75 numerically coded independent variables can be obtained from the authors . the parameters of the mcmc experiment ( e.g. , burn - in period , thinning rate , etc . ) the estimated qtl effects and the permutation - generated 2.5%97.5% and 5%95% intervals are plotted in figure 10(a ) . when we lowered the critical value to = 0.10 , one more qtl was detected on chromosome 5 . the cross - validation shows that the optimal strategy of genome selection for this dataset was to include all qtls that are significant at = 0.1 ( see figure 10(b ) ) . below = 0.1 , the decrease of pe was dramatic but after = 0.1 the pe values increased slightly until they reached a plateau at = 0.3 . in general , the optimal gamma value is somewhere between 0.1 to 0.2 , but it varied from one experiment to another . the last two data analyses did indicate that including small qtl can be detrimental to genome selection . cross - validation is an experimental specific approach and is useful to decide how large a qtl should be included in the model for genome selection . qtl mapping aims to detecting qtl with large effects while genome selection tries to predict the total genetic values of individuals using markers of the entire genome . in qtl mapping , significance test is important , but bayesian inference usually does not mix with significance test . this is because bayesian inference focuses on the probability statement of a parameter given the information drawn from the current data and it does not intend to extend the statement beyond the data . significance test , however , assumes a null distribution and tries to compare the statistics against the null distribution . the null distribution is purely hypothetical and , therefore , significance test gives conclusion that applies to hypothetical future experiments . the permutation test adopted in the bayesian analysis is a convenient way to connect significance test with bayesian analysis . permutation analysis is a way to draw the null distribution . if a statistics , for example , estimated qtl effect , is far away from the null distribution , we are confident that this qtl is true . this type of significance test provides different conclusion from the bayesian credible statement . in bayesian analysis , people often report the -equal - tail interval or -highest posterior density ( hpd ) interval . the reason is that almost all qtls have an equal - tail interval covering the null value , for example , zero . even the largest qtl in our simulation had a high probability mass at zero ( see figure 2 ) . this zero - inflated posterior distribution for qtl effect is typical in bayesian shrinkage mapping . if we had used the equal tail interval at = 0.05 as the significance test criterion , only one qtl ( the largest one ) , out of the 20 simulated qtls , would have reached the statistical significance level . in the simulation experiment , we observed that the percentile profiles for the 0.5 100% ( 1 0.5 ) 100% interval were pretty much constant across the entire genome ( see figures 3 and 4 ) . we simulated 241 markers covering the entire genome evenly with 10 cm per marker interval . in contrast , the three real data analyses showed that the percentile lines varied dramatically across the genome . the intervals were narrow at marker positions and wide when the positions are away from the markers . the lengths of marker intervals also varied across the genome , making the information content much uneven across the genome . the location specific empirical threshold values in real data analysis mean that different locations of the genome should use different criteria for qtl detection . we have two qtl with the same estimated effect but located in different regions of the genome , one may be declared as significant but the other may not be significant due to the variation in information content . this actually justifies the use of estimated qtl effects , not some kind of test statistics , for significance test . in classical qtl mapping experiments , investigators always use some kinds of test statistics ( e.g. , t - test , f - test , likelihood ratio test , or lod score ) to decide whether a qtl is significant or not . a permutation test also draws critical values for the test statistic under consideration , not the critical values for the qtl effects . this merely reflects the tradition or convention of people who do statistical analysis and does not mean that a test statistic is the only quantity that can be used in qtl mapping . the reason for using test statistics is that one can compare the observed test statistic ( calculated values ) with the critical values of some distribution , for example , normal distribution , f - distribution , t - distribution , and chi - square distribution . the critical values of these standard distributions can be found from statistical tables or calculated from statistical analysis software . with the permutation test therefore , there is no need to use the test statistics . directly comparing the estimated qtl effects with the critical values the significant qtl will be the targets for further study , for example , cloning or marker assisted selection . what do we do with those qtls whose effects do not reach the significance level ? these qtls may not be significant individually , but collectively they may contribute to a large proportion of the phenotypic variance . this implies that they are perhaps useful to predict the total genetic effects of individuals , a technology called genome selection . our cross - validation experiments showed that qtls should be used to predict the total genetic effects once they reached a certain critical value . common sense tells us that estimated effects of small qtls are most likely caused by noises rather than by true signals and inclusion of the many small qtls to predict the genetic effects may be even worse than inclusion of only the significant qtl . we developed two permutation tests , one is called permutation outside markov chain and the other is called permutation inside markov chain . the latter is recommended because it is ( 1 ) faster computationally and ( 2 ) slightly more conservative . cross - validation studies showed that small qtls should be excluded from the model for prediction of the total genetic effects for individual plants . the criterion of exclusion is experimental specific and should be decided by cross - validation .

bayesian shrinkage analysis is the state - of - the - art method for whole genome analysis of quantitative traits . it can estimate the genetic effects for the entire genome using a dense marker map . the technique is now called genome selection . a nice property of the shrinkage analysis is that it can estimate effects of qtl as small as explaining 2% of the phenotypic variance in a typical sample size of 300500 individuals . in most cases , qtl can be detected with simple visual inspection of the entire genome for the effect because the false positive rate is low . as a bayesian method , no significance test is needed . however , it is still desirable to put some confidences on the estimated qtl effects . we proposed to use the permutation test to draw empirical thresholds to declare significance of qtl under a predetermined genome wide type i error . with the permutation test , bayesian shrinkage analysis can be routinely used for qtl detection .

1. Introduction 2. Methods 3. Results and Discussion 4. Discussion 5. Conclusions

PMC3395935

a sample of 103 voluntary male participants , based on their response to the following question from the whoqol - bref questionnaire : are you currently ill ? ( yes - no ) , were divided into two groups : unhealthy ( n=55 ) and healthy ( n=48 ) respectively . whoqol - bref and the depression anxiety stress scale ( dass-42 ) were used to assess satisfaction rating and negative emotions of depression , anxiety and stress . the who 's qol scale ( whoqol - bref ) was used to assess satisfaction with four domains of life : physical health , psychological , social relationships and environment support . the four domain scores are scaled in a positive direction , with a score range of 0 - 20 , and with higher scores denoting higher satisfaction . the 42-item format of dass was used to measure the negative emotional states of depression , anxiety and stress . in order to find how rating the whoqol - bref and dass scales is combined to produce an overall measure of quality of life and satisfaction with health rating , a qol - dass model was designed ; and the strength of this hypothesized model of qol - dass was examined using structural equation modeling ( figure 1 ) . in this model , ellipses represent latent variables ; whoqol , and dass , and rectangles represent measured variables ; satisfaction in four different domains of life , physical health , psychological well - being , social relations , and environment , as measured with whoqol - bref and depression , anxiety , stress as measured with dass-42 . a sample of 103 voluntary male participants , based on their response to the following question from the whoqol - bref questionnaire : are you currently ill ? ( yes - no ) , were divided into two groups : unhealthy ( n=55 ) and healthy ( n=48 ) respectively . whoqol - bref and the depression anxiety stress scale ( dass-42 ) were used to assess satisfaction rating and negative emotions of depression , anxiety and stress . the who 's qol scale ( whoqol - bref ) was used to assess satisfaction with four domains of life : physical health , psychological , social relationships and environment support . the four domain scores are scaled in a positive direction , with a score range of 0 - 20 , and with higher scores denoting higher satisfaction . the 42-item format of dass was used to measure the negative emotional states of depression , anxiety and stress . in order to find how rating the whoqol - bref and dass scales is combined to produce an overall measure of quality of life and satisfaction with health rating , a qol - dass model was designed ; and the strength of this hypothesized model of qol - dass was examined using structural equation modeling ( figure 1 ) . in this model , ellipses represent latent variables ; whoqol , and dass , and rectangles represent measured variables ; satisfaction in four different domains of life , physical health , psychological well - being , social relations , and environment , as measured with whoqol - bref and depression , anxiety , stress as measured with dass-42 . for analysis , a confirmatory factor analysis ( cfa),using maximum likelihood , was conducted on a sample of 103 participants whose scores derived from two rating scales of whoqol - bref and dass-42 . using amos and maximum likelihood estimation , the relationships were examined between the whoqol , a latent variable with 4 indicators of four domains concerning with satisfaction with physical health ( dom1 ) , psychological well - being ( dom2 ) , social relationships ( dom3 ) and environment support ( dom4);and another latent variable , dass , with 3 indicators of depression , anxiety , and stress ratings . the most commonly used goodness - of - life index is the chi - square statistic . the results of the run of the data obtained from the proposed model of qol - dass is presented in table 1 . fit indices of the model of qol - dass for different healthy and unhealthy groups = chi - square ; df= degrees of freedom ; cfi= comparative fit index ; nnfi= non - normed fit index ; nfi= normed fit index ; rmsea = root mean square error of approximation ; pclose = probability of close fit as observed in table 1 , for both the healthy and unhealthy groups , the 2 statistic for the proposed model of qol - dass indicates that the proposed model fit the data well , which seemed to suggest an inadequate fit of the model . moreover , other indicators indicated much more favorable results . table 2 provides estimated regression weights ( b ) and standardized regression weight ( ) coefficients of all pathways for the hypothesized model of qol - dass . regression weights ( b ) and standardized regression weight ( ) coefficients in order to find whether or not components of the measurement model ( qol - dass model ) are invariant across particular groups ( here scores are derived from healthy and unhealthy persons),a multiple group analysis was conducted on the two groups . these groups varied depending on their response to a question ( do you feel ill now ?- the no response for the healthy group ; and the yes response for the unhealthy group . the question is whether the relation between specific items ( satisfaction with physical health ( dom1 ) , psychological well - being ( dom2 ) , social relationships ( dom3 ) , environment support ( dom4 ) , depression , anxiety , and stress ratings . ) and the underlying construct or factor ( whoqol and dass ) tapped by the item is the same across groups . unconstrained model does not specify any constraints on groups of healthy and unhealthy participants , and the measurement weights model specifies all the factor loadings as equal on the different studied groups . fit indices as well as the change in 2 , and change in degrees of freedom between unconstrained and measurement weights models are presented in table 3 . goodness - of - fit statistics for tests of invariance across different groups cfi= comparative fit index ; rmsea = root mean square error of approximation as demonstrated in table 3 , the comparison of models , unconstrained and measurement weights , was found to be non - significant across healthy / unhealthy groups ( 2 = 7.95 , df = 9 , ns . given this finding , it can be concluded that the factor loading is shown invariant across the studied groups . in order to find whether or not components of the measurement model ( qol - dass model ) are invariant across particular groups ( here scores are derived from healthy and unhealthy persons),a multiple group analysis was conducted on the two groups . these groups varied depending on their response to a question ( do you feel ill now ?- the no response for the healthy group ; and the yes response for the unhealthy group . the question is whether the relation between specific items ( satisfaction with physical health ( dom1 ) , psychological well - being ( dom2 ) , social relationships ( dom3 ) , environment support ( dom4 ) , depression , anxiety , and stress ratings . ) and the underlying construct or factor ( whoqol and dass ) tapped by the item is the same across groups . unconstrained model does not specify any constraints on groups of healthy and unhealthy participants , and the measurement weights model specifies all the factor loadings as equal on the different studied groups . fit indices as well as the change in 2 , and change in degrees of freedom between unconstrained and measurement weights models are presented in table 3 . goodness - of - fit statistics for tests of invariance across different groups cfi= comparative fit index ; rmsea = root mean square error of approximation as demonstrated in table 3 , the comparison of models , unconstrained and measurement weights , was found to be non - significant across healthy / unhealthy groups ( 2 = 7.95 , df = 9 , ns . given this finding , it can be concluded that the factor loading is shown invariant across the studied groups . george & bearon , ( 16 ) , define quality of life in terms of four underlying dimensions , two of which are objective ( general health and functional status and socioeconomic status ) and two of which reflect the personal judgment of the individual ( life satisfaction and related measures and self - esteem and related measures ) , and believe that these dimensions are especially central for assessment of quality of life of older people ( 16 ) . although the boundary between subjective and objective components of qol is not always clear in practice , measures of quality of life are based on both objective and subjective variables ( 17 ) . meeberg ( 1 ) referring to subjective and objective components of quality of life , claimed that the subjective aspect is essential because a sense of personal satisfaction is intrinsic to qol and the objective component is also necessary . a person living in poverty and squalor who has never known any other way of life may feel satisfied with his or her life . yet , a person from outside those living conditions can see the prevailing health hazards and would evaluate that individual 's qol as less than ideal ( 1 ) . in order to find how subjective evaluation of satisfaction with life domains and feeling negative emotions affect perceived general health and quality of life , a hypothesized model of qol - dass was designed , and the strength of this hypothesized model was examined using structural equation modeling ( see fig . our results of running the hypothesized model of qol - dass indicated that the proposed model of qol - dass fitted the data well for both healthy and unhealthy groups ( see table 1 ) . we continued our analysis by testing for multiple group invariance to find whether or not components of the measurement model , qol - dass model , are invariant across the two groups . our results of conducting multiple group invariance across the groups of healthy and unhealthy indicate that all factor loadings across different groups used to be invariant ( table 3 ) . as the baseline ( unconstrained ) and regression weights models are not significantly different , it is concluded that the hypothesized model of qol - dass is invariant between the two groups . our findings to evaluate the hypothesized model of qol - dass indicate that the different satisfaction domain ratings obtained from whoqol - bref and the negative emotions ( depression , anxiety and stress measured using dass-42 as the observed variables ) can represent the underlying constructs of general health and quality of life on both healthy and unhealthy groups .

objectivein order to find how rating the whoqol - bref and dass scales are combined to produce an overall measure of quality of life and satisfaction with health rating , a qol - dass model was designed ; and the strength of this hypothesized model was examined using the structural equation modeling.methodparticipants included a sample of 103 voluntary males who were divided into two groups of unhealthy ( n=55 ) and healthy ( n=48 ) . to assess satisfaction and negative emotions of depression , anxiety and stress among the participants , they were asked to fill out the whoqol - bref and the depression anxiety stress scale ( dass-42).resultsour findings on running the hypothesized model of qol - dass indicated that the proposed model of qol - dass fitted the data well for the both healthy and unhealthy groups.conclusionour findings with cfa to evaluate the hypothesized model of qol - dass indicated that the different satisfaction domain ratings and the negative emotions of depression , anxiety and stress as the observed variables can represent the underlying constructs of general health and quality of life on both healthy and unhealthy groups .

Materials and Method Participants Instruments Statistical Analyses Results Testing for multiple group invariance Discussion

PMC4279838

a 37-year - old man was admitted to our hospital with long - standing , persistent , lone atrial fibrillation refractory to medical therapy and several attempts of cardioversion . the patient underwent a total thoracoscopic epicardial ablation without any intraoperative or postoperative complications , followed by a percutaneous postprocedural electrophysiologic evaluation that included confirmation of pulmonary vein isolation and a cavotricuspid isthmus block on the tenth postoperative day . two weeks after discharge , the patient was admitted to the emergency room with right - sided motor weakness and paresthesia of both upper and lower extremities . his fever reached 40c , and he had a white blood cell count of 17,690/l . chest computed tomography imaging revealed a collection of loculated air measuring up to 2 cm in diameter between the right side of the left atrium and the esophagus , with no definitive evidence of a fistulous connection ( fig . 1 ) . brain computed tomography revealed an acute - onset left middle cerebral artery infarction . an urgent surgical intervention was performed due to our high level of suspicion for a left atrial or pulmonary venous - esophageal fistula . the chest was opened through the fifth intercostal space via a right posterolateral thoracotomy under one - lung ventilation with a double lumen endotracheal tube . after anteriorly retracting the lung , a meticulous dissection of the posterior mediastinum between the esophagus , right upper and lower pulmonary veins , and left atrium was performed . a fistula 2 to 3 mm in diameter was discovered between the right lower pulmonary vein and anterior surface of the esophagus . surprisingly , there was no bleeding from the fistula on the side of the right lower pulmonary vein . the right lower pulmonary vein was repaired with a bovine pericardial patch , covered , and fixed with continuous poly - propylene sutures . after repairing the right lower pulmonary vein , we consulted our hospital s thoracic surgeon about repairing the fistula opening on the esophageal side . he decided to carry out a primary repair of this defect because the defect site was small and relatively clear . the fistula was carefully debrided and repaired with intermittent 4 - 0 black silk sutures . the patient s rhythm was converted to sinus rhythm and sustained after cardioversion one day after surgery . the patient became alert on the third postoperative day , and was extubated on the fifth postoperative day . he was discharged 39 days postoperatively , with a motor power score of three out of five on the medical research council scale after rehabilitation therapy . on his last outpatient visit , two and a half months after surgery , motor power was fully recovered without any sequelae . a hybrid procedure of a total thoracoscopic epicardial ablation and percutaneous transvenous radiofrequency catheter ablation for lone atrial fibrillation , performed either simultaneously as a single stage procedure or as a two - stage procedure , has been developed to overcome the limitations of each technique and result in better outcomes . the success rate of this therapy varies according to the specific goals of the intervention and the duration of atrial fibrillation ( paroxysmal or persistent ) . pison et al . reported conversion to sinus rhythm after a single stage procedure with a success rate of 83% after one year . muneretto et al . reported conversion to sinus rhythm after a two - stage procedure with a success rate of 91.6% at a mean follow - up of 30 months . after being performed for the first time in south korea at the samsung medical center in february 2012 , we have performed 50 staged hybrid procedures to date with a successful conversion to sinus rhythm in 48 out of 50 patients . unfortunately , we experienced a potentially fatal case of a fistula running from the pulmonary vein to the esophagus fistula despite the high success rate of conversion to sinus rhythm . we believe our case of a right lower pulmonary venous - esophageal fistula after thoracoscopic epicardial and transvenous catheter ablation to be the first reported in the literature . even though the radiofrequency catheter ablation was performed after the total thoracoscopic epicardial ablation in a staged procedure , we think that there may have been some degree of infection or edema at the ablation sites due to the short ( ten days ) interval between the components of the hybrid procedure . the most commonly reported esophageal complication after radiofrequency catheter ablation are left atrial - esophageal fistulas , with an incidence rate of up to 0.2% . these fistulas result from the usual anatomical relationship of the esophagus to the left atrium and pulmonary veins . however , unusual variations where the esophagus is situated rightward in the posterior mediastinum near the right pulmonary veins have been described , as in our present case . . a median sternotomy or a right or left thoracotomy with or without cardiopulmonary bypass is possible depending on the clinical situation and location of the fistula . surgical exposure in our case was achieved via a right posterolateral thoracotomy to expose the suspicious right pulmonary veins and left atrium . in light of this rare but potentially lethal event , we suggest the development of a better - defined consensus , based on further studies , on whether hybrid thoracoscopic epicardial and percutaneous catheter ablations should be performed simultaneously or as a staged procedure .

a case of a fistula running from the pulmonary vein to the esophagus after a staged hybrid procedure combining total thoracoscopic ablation and percutaneous radiofrequency catheter ablation has not been reported previously . we describe such a case in a 37-year - old man who was successfully treated by surgery .

CASE REPORT DISCUSSION

PMC4842059

the circadian clock is an autoregulatory network that regulates behavioral and metabolic programing in the context of a 24 h light - dark ( ld ) cycle . body temperature is one of the representing benchmarks of circadian patterning , which peaks in animals while awake and troughs while asleep . brown adipose tissue ( bat ) is a major site for rodent thermogenesis , due to its involvement in controlling circadian thermogenic rhythms and influencing adaptability to environmental temperature challenges . a previous study has revealed rhythmic expression patterns of over 5,000 genes in murine bat , including genes associated with the circadian clock , adipose function , and metabolism . moreover , the inhibitor of dna binding 2 ( id2 ) gene encodes a helix - loop - helix ( hlh ) transcriptional regulator , which is rhythmically expressed in many mammalian tissues and involved in the input pathway , core clock function , and output pathways of the circadian clock [ 69 ] . our previous studies have shown that id2/ mice exhibit lower levels of locomotor activity , extended nighttime activity patterns of feeding and locomotor activity , and sex- and age - dependent enhanced glucose tolerance and insulin sensitivity . moreover , an energy - rich diet is able to rescue the disturbances to metabolic homeostasis and survival in the id2/ mice sex - specifically . importantly , id2/ mice show a sex - dependent elevated glucose uptake in interscapular bat ( ibat ) . id2 also plays a role in white adipose tissue ( wat ) adipogenesis [ 1012 ] . however , the role of id2 on temperature homeostasis regulation and its influence on bat physiology remain unknown . therefore , we investigated the function of id2 in the regulation of temperature rhythms under normal and thermoneutral conditions in a sex - specific manner and also profiled the expression of genes involved in insulin signaling and adipogenesis in bat of id2/ mice , sex - specifically . the generation of id2/ mice and genotype determination were performed as described previously [ 7 , 10 , 11 ] . mice were maintained on a regular chow diet and sterile water containing antibiotic ad libitum [ 7 , 10 , 11 ] . all mice were housed in laboratory cages at normal temperature ( 21c 1c ) and humidity of 5065% under a 12 : 12 light : dark ( ld ) cycle with lights on at zeitgeber time ( zt ) 0 and lights off at zt12 . animal experiments were approved by the university of notre dame animal care and use committee ( protocol number 14 - 02 - 1559 ) and performed in accordance with nih guidelines for the care and use of laboratory animals . temperature measurements were carried out on 2-month1.5-year- ( 5.5-month median ) old male and female id2/ mice and wt littermates , housed individually in a climate - controlled room set to either normal ( 21c 1c ) or thermoneutral ( 30c 1c ) temperature . body temperature sampling was conducted at 3 h intervals over the 24 h ld cycle . for thermoneutral conditions measurement , all wt and id2/ mice used in the studies were allowed to acclimate to thermoneutral temperature for 1 week before temperature measurement . core body temperature was measured using subcutaneously surgically implanted telemetric transmitters positioned proximal to the ibat ( iptt 300 transponders , bio medic data systems , seaford , de ) following isoflurane anesthetization . after a week of recuperation , ibat tissue was harvested at zt16 ( id2 mrna circadian rhythm in ibat has a broad peak phase between zt8 and zt16 ) . id2/ and wt male ( wt = 8 , id2/ = 6 ) and female ( wt = 6 , id2/ = 4 ) mice from 39 months ( 6.1-month median ) were sacrificed and ibat tissue was frozen in liquid nitrogen and stored at 80c until analyzed . we also measured ibat weight of these and additional mice ( 310-month - old id2/ mice and wt littermates ; 6.3-month median ; male , wt = 15 , id2/ = 7 ; female , wt = 10 , id2/ = 9 ) as described previously [ 10 , 11 ] . rna integrity was assessed using a bioanalyzer 2100 ( agilent technologies , santa clara , ca ) . rna was subjected to a dnase i treatment , and cdna was synthesized by rt first strand kit ( sabiosciences ) . relative mrna expression of 168 genes involved in insulin signaling and adipogenesis pathways was determined by using the mouse pcr arrays ( pamm-030zc-24 and pamm-049zc-24 , sabiosciences ) . quantitative real - time pcr was performed using an applied biosystems 7500 system with rt2 sybr green rox qpcr master mix reagent ( qiagen ) . pcr array data were calculated by the comparative cycle threshold method and analyzed by web - based free pcr array data analysis software provided by sabiosciences . normalization of expression was to housekeeping genes provided on each array ( actb , b2 m , gapdh , gusb , and hsp90ab1 ) . clock controlled genes ( ccgs ) were identified from the circa database of mouse 1.ost brown adipose ( affymetrix ) ( http://bioinf.itmat.upenn.edu/circa/ ) where we defined ccgs as a jtk_cycle algorithm determined q < 0.1 value and a period length of 2028 h as described previously [ 1315 ] . the linear regression of ibat temperature - body weight relationship was generated and analyzed using prism 5.0 graphpad software . pcr array data were analyzed using the web - based free pcr array data analysis software provided by sabiosciences ( student 's t - test ) . the discovery of a diurnal rhythm of glucose uptake in mice ibat and a sex - dependent elevated glucose uptake in ibat of id2/ mice prompted us to investigate whether id2 contributes to thermoregulation [ 5 , 10 ] . at normal ambient temperature conditions ( 21c ) , ablation of id2 reduced core body temperature across the 24 h day , in both male and female mice ( figure 1(a ) ) ( males , wild types ( wts ) = 14 , id2/ = 14 , anova , time ( t ) , p < 0.001 , genotype ( g ) p < 0.001 , interaction ( i ) , n.s . ; females , wts = 18 , id2/ = 17 , anova , t , p < 0.001 , g , p < 0.05 , i , n.s . ) . considering the possibility of any confounding genetic background contribution and partial stimulation of bat activity occurring under normal temperature conditions , id2/ mice core body temperature was also measured under thermoneutral conditions ( 30c ) [ 3 , 16 ] . consistently , at thermoneutrality , id2/ mice displayed a reduced core body temperature ( figure 1(a ) ) ( males , wts = 19 , id2/ = 20 , anova , t , p < 0.001 , g , p < 0.01 , i , n.s . ; females , wts = 18 , id2/ = 17 , anova , t , p < 0.001 , g , p < 0.01 , i , n.s . ) . under both conditions and in both sexes , no interaction between time and genotype was discovered , suggesting a generalized effect of the null mutation on core body temperature rather than a time - of - day specific contribution of the gene deletion . regression analysis of time - of - day representative core body temperatures ( day or night ) revealed no significant relationships between temperature and body mass for either id2/ or wt mice . however , id2/ mice of both sexes showed consistently lower y - intercept lines compared to wt mice when examined during either the daytime ( zt5.5 ) or nighttime ( zt17.5 or zt20.5 ) , thus confirming the consistently lower temperature of the id2 null mice ( figure 1(b ) ; supplementary table 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/6785948 ) . lastly , id2/ mice exhibited no statistically significant difference in ibat weight and ibat to body weight ratio compared to wt controls ( figures 2(a ) and 2(b ) ) ( two - factor anovas , ibat weight , g , n.s . , sex ( s ) , p < 0.01 , i , n.s . ; ibat / body weight ratio , g , n.s . , s , n.s . the mean and sem body mass of wt and id2/ mice for both ibat weight and body temperature experiments are shown in supplementary table 2 : note that both male and female id2/ mice had on average a lower body mass compared to wt counterparts ( two - factor anova , g , p < 0.001 , s , p < 0.001 , i , n.s . ) . our previous results showed sex - dependent enhanced insulin sensitivity and glucose uptake in ibat of id2/ mice . in the current study we observed a decreased core body temperature in id2/ mice as described above . to fully evaluate the impact of ablation of id2 on bat gene - regulation , we performed a gene expression analysis using rt profiler pcr arrays of bat derived from id2/ mice and their wt littermates collected at the same time of the 24 h day ( specifically zt16 ) . deferentially regulated genes involved in insulin signaling and adipogenesis are shown in tables 1 and 2 , respectively . thirty of 168 genes examined were identified as differentially expressed when analyzed as a cohort or as individual sex - specific groups . using the circa database as a resource , six genes were identified as clock controlled genes ( ccgs ) , of which four oscillate in proximal phase with the rhythm of peroxisome proliferator activated receptor alpha ( ppar ) , peaking during the middle of the day ( ~circadian time ( ct ) 6 ; ct12 = onset of night in prior ld cycle ) . of importance for insulin signaling , glucose-6-phosphatase , catalytic ( g6pc ) , was upregulated in id2/ females and the related g6pc family member g6pc2 downregulated in id2/ males ( p = 0.079 , approaching significance ) compared to wts . insulin receptor substrate 1 ( irs1 ) was upregulated in both male and female id2/ mice . insulin - like growth factor 2 ( igf2 ) was downregulated in female id2/ mice , while fbp1 , a rate - limiting enzyme in gluconeogenesis , and shc1 , a component in the igf-1-regulated pathway , were upregulated . for adipogenesis , bone morphogenetic protein 4 ( bmp4 ) was elevated 1.7-fold ( n.s . ) in male and 1.6-fold in female id2/ mice . nuclear receptor coactivator 2 ( ncoa2 ) , pr domain containing 16 ( prdm16 ) , ppar , and twist homolog 1 ( twist1 ) were downregulated , in grouped analysis of male and female id2/ mice . fatty acid synthase ( fasn ) , lipase , hormone sensitive ( lipe ) , and peroxisome proliferative activated receptor , gamma , coactivator 1 beta ( ppargc1/pgc-1 ) were all downregulated in male id2/ mice . female id2/ mice displayed a downregulation of proliferative activated receptor , gamma , coactivator 1 alpha ( ppargc1/pgc-1 ) . a small 1.2-fold downregulation of peroxisome proliferator activated receptor gamma ( ppar ) was detected in id2/ males , where the p value was approaching significance ( p = 0.061 ) . note that the thermogenic protein , uncoupling protein 1 ( ucp1 ) , was present on both the insulin signaling and adipogenesis arrays , but its levels of expression were not significantly altered in the id2/ mice . in the present study , we discovered a reduced core body temperature in id2/ mice , and this effect was not found to be dependent upon the time - of - day . moreover , from the ibat of id2/ mice , genes involved in insulin signaling and adipogenesis were differentially regulated in a sex - dependent manner . these results reveal a role of id2 in the regulation of thermogenesis and bat metabolic functions . our previous study revealed that id2/ mice exhibit less activity as demonstrated by daily counts of general activity and the wheel running activity , which could partially explain the reduced core body temperature , since less physical activity would generate less heat . moreover , id2/ mice show a reduced body mass and less gonadal adipose deposits [ 6 , 10 , 11 ] . as the subcutaneous and intradermal fat functions as thermal insulation for mice to preserve heat loss , id2/ mice with low fat content might tend to lose heat more readily than wt mice . furthermore , the reduced body temperature associated with lower fat content might contribute to the high death rate observed previously ( mice housed under normal temperature ) , which was rescued by a high fat diet that resulted in increased total body fat . specifically in male id2/ mice ibat we observed increased glucose uptake and reduced tg accumulation , suggesting alterations in its metabolic programing . interestingly , our results suggest that the role of id2 in thermoregulation is opposite to the function of another member of this hlh family , id1 , whose deficiency results in higher thermogenesis and an elevated bat expression of thermogenic proteins . notably , id1 has a distinct and opposite function in wat adipogenesis compared to id2 , despite both id1 and id2 null mice exhibiting reduced adiposity [ 1012 , 17 ] . lastly , we examined the relationship between body mass and body temperature in id2/ mice by regression analysis and revealed a limited relationship between the two variables . no significant relationship was observed between body mass and body temperature at any time of the day or in the two sexes . however , as can be seen with the y - intercept of the regression lines , both id2/ male and female mice expressed a consistently lower temperature compared to wt controls , irrespective of body mass , and this feature was observed during both the day and night phases of the ld cycle . these results suggest a role for id2 in the regulation of core body temperature . in this study we also measured ibat mass and ibat / body mass ratio . while there was a tendency for higher ibat / body mass in both id2/ male and female mice , this was not determined to be a significant difference . note that the average body mass of id2/ mice used for both the ibat weight and body temperature experiments was found to be significantly lower , consistent with our previous studies [ 7 , 10 ] . important is the fact that a lower body mass , found for some of the id2/ mice and for males in particular , does not correlate with a lower body temperature , and body mass in this situation is therefore an independent factor when predicting core body temperature . it is important to note that while the objective of examining body temperature using the implanted thermometers was to record core body temperature , the position of the implants may not give an exact measure of true core body temperature . however , in a comparable study of mouse body temperatures , temperature measurements were similar whether derived from similarly subcutaneously implanted thermometers in the interscapular region of wt and rev - erb mutant mice or as determined using dataloggers that were implanted within the abdomen . id2 is rhythmically expressed in bat [ 4 , 15 ] amongst other tissues [ 6 , 7 ] . id2 protein has also been observed to be rhythmic in its abundance over the 24 hr diurnal / circadian cycle within the liver and heart ( ward , fernando , hou , and duffield , unpublished data ) . a role for id2 has been established as a mediator of circadian clock output and control of expression patterns of clock controlled genes ( ccgs ) within the liver . it is for this reason that we examined whether any of the genes identified as differentially expressed in ibat were in fact known ccgs . using the circa database [ 14 , 15 ] , 5 of the 17 differentially genes associated with adipogenesis were found to be ccgs ( e.g. , ppar and pgc-1 ) , and so a possible role for id2 is in mediating circadian regulatory effects on these genes within bat . the observation that few of the differentially regulated genes involved in insulin signaling are ccgs ( 1 out of 13 genes ) suggests that the contribution of id2 to insulin signaling intrinsic to bat is independent of the role of id2 in mediating circadian clock output . in order to explain how id2 deficiency has an impact on bat insulin signaling and adipogenesis the nuclear receptor ppars are fundamentally important for energy homeostasis and id2 plays a role in interfacing with the molecular pathways upstream or downstream of these transcriptional factors . expression of two members of the ppar subfamily of ligand - activated nuclear receptors , ppar and ppar , was downregulated in our study . ppar is highly expressed in bat and considered a marker of bat ; it also plays an important role in the overall regulation of lipid metabolism ; and its target genes are involved in mitochondrial and peroxisomal -oxidation of fatty acids ( fas ) [ 2022 ] . moreover , ppar regulates the expression of uncoupling protein 1 ( ucp1 ) , which confers on bat its thermogenic capacity . pgc-1 ( downregulated in our study ) is a transcriptional coactivator involved in the control of energy metabolism and critical for bat thermogenesis and enhancing overall mitochondrial oxidative activity . ppar can induce pgc-1 gene expression and contributes to the thermogenic activation of brown fat . prdm16 exhibits a brown fat selective expression pattern and regulates the thermogenic gene program in brown and beige adipocytes . the observation of reduced prdm16 expression in id2/ mice is consistent with the role of prdm16 as a transcriptional regulator of pgc-1 . ppar is essential for adipocyte differentiation , and ppar alone generates a fat phenotype that is common to both wat and bat . the ccaat enhancer binding protein beta ( c / ebp ) and pgc-1 are critical for controlling ppar expression in bat and for determining bat - specific programs [ 29 , 30 ] . the ppar thermogenic effect in bat it has been observed that overexpression of id2 associates with ppar expression , id2 acts upstream of ppar , and c / ebp induces id2 expression during the adipogenesis process [ 12 , 31 ] . cofactors such as ncoa2 ( downregulated in our study ) can interact directly with ppar to initiate its own transactivation . moreover , lipe ( downregulated in our study ) could modulate adipose metabolism by reducing the availability of ligands for ppar , since gene knockout of lipe in mice attenuates activation of ppar . lipe is also able to hydrolyze stored tgs in adipose tissue and to mobilize free fa from adipose tissue . furthermore , ppar is a direct target of the transcription factor sterol response element binding protein 1 ( srebp1 ) , whose transcriptional activity is modulated by id2 and which regulates downstream lipid metabolism genes such as lipe and fasn [ 35 , 36 ] . additionally , irs1 ( upregulated in our study ) plays essential roles in the differentiation of brown adipocytes and expression of ppar [ 37 , 38 ] . previous studies have revealed irs1-regulated id2 gene expression , although in the current study it is unclear whether this is a direct effect or a feedback response . as for the mechanism by which irs1 is elevated in id2/ ibat , bmp4 ( upregulated in our study ) is able to induce the white to brown transition of adipose cells , which could indirectly regulate ppar activation [ 40 , 41 ] . the elevated bmp4 expression in the context of reduced ppars is surprising since bmp4 upregulation is associated with increased bat adipogenesis and the wat browning effect . interestingly , the id2 gene promoter has bmp - response elements and has been shown to be a target of bmp signaling . ppar plays an integral role in transcriptional network regulation of fat - burning genes and brown fat metabolism . twist-1 ( downregulation in our study ) encodes a basic hlh transcription factor , and overexpression of twist-1 is associated with id2 expression . note that the gene encoding the thermogenic protein , ucp1 , was present on the pcr arrays , but no difference in its expression was observed between genotypes of either sex . interestingly , in the id1 null mouse , ucp1 gene expression is elevated in ibat , and this is associated with an increased core body temperature phenotype . thus , it is surprising that in the id2/ mouse that exhibits a reduced body temperature phenotype we do not observe a reduction in ucp1 gene expression . of course , this does not exclude , however unlikely , the possibility of an altered ucp1 protein abundance through a posttranscriptional / posttranslational process . hypoxia , while not a focus of the current study , is known to reduce body temperature in mammals and contribute to the thermogenic activity of bat . it is noteworthy that in a recent study of human glioblastoma cells / tissue , an important role was established for id2 in modulating the cellular effects of hypoxia and its activation of the hif2 pathway . the id2 gene is also a target for hif1 and hif2 [ 47 , 48 ] , making it part of a positive feedback loop mechanism , at least in models of brain tumor . it is plausible that the hypoxic effects on bat function might also include a contribution from id2 , and this would be an important pathway to examine in future experiments in this context . it is a somewhat contradictory finding that elements of the thermogenic pathway are reduced ( e.g. , pgc1- ) or unaltered ( ucp1 ) in id2/ mice but that id2/ male ibat exhibits increased glucose uptake ( pet - fdg ) coupled with reduced ibat triglyceride levels and a systemwide enhanced insulin sensitivity [ 10 , 11 ] and that core body temperature is consistently reduced in both id2/ male and female mice . id2/ mice also exhibit an altered 24 hr locomotor activity and feeding profile and an overall reduction in nocturnal locomotor activity , the latter of which suggests a reduction in energy expenditure / increased energy conservation . clearly the bat adipogenic program is altered in both male and female id2/ mice , as is wat adipogenesis [ 1012 ] , and it is likely that a change in bat function contributes to the reduced temperature phenotype . due to the nature of whole body knockout of id2 , it is possible that the temperature phenotype and ibat gene changes observed are secondary to whole body metabolic changes . clearly additional experiments are required to elucidate the relative contributions of these and other potential components in generating the altered core body temperature phenotype . our previous study showed enhanced glucose uptake in ibat of id2/ mice , and the present study reveals a reduced core body temperature in id2/ mice . this discrepancy could partially be explained by the differential regulation of irs1 , lipe , ppars , and pgc-1s . it has been reported that degradation of irs1 leads to impaired glucose uptake in adipose tissue . therefore , upregulation of irs1 might explain the increased glucose uptake we observed before , whereas downregulation of lipe , ppars , and pgc-1s might contribute to reduced fa oxidation , impaired adipogenesis , and a lower body temperature . inactivation of ppars is associated with insulin resistance [ 50 , 51 ] , yet paradoxically id2/ mice show enhanced insulin sensitivity with downregulated ppars [ 10 , 11 ] . it has been suggested that mice that lack one allele of the ppar gene are more sensitive to insulin , which could partially explain the enhanced insulin sensitivity we observe in id2 null mice [ 10 , 51 ] . furthermore , the differential regulation of genes specifically in female mutant mice , such as fbp1 , a rate - limiting enzyme in gluconeogenesis , shc1 , a component in the igf-1-regulated pathway , and igf2 , suggests a sex - specific physiological program for id2 in bat . inhibitor of dna binding 2 is rhythmically expressed in bat [ 4 , 15 ] , and the observation that few of the differentially regulated genes involved in insulin signaling are ccgs suggests that the contribution of id2 to insulin signaling intrinsic to bat is independent of the role of id2 in mediating circadian clock output . overall , id2 seems to be an important coordinator of energy homeostasis including insulin signaling , adipogenic programing , and thermoregulation . in conclusion , our finding that id2 contributes to the regulation of body temperature and energy homeostasis presents the possibility that id2 could be a potential therapeutic target for metabolic disease . further , these data emphasize the influence of id2 on bat molecular signaling and physiology in a sex - specific manner .

inhibitor of dna binding 2 ( id2 ) is a helix - loop - helix transcriptional repressor rhythmically expressed in many adult tissues . our previous studies have demonstrated that id2 null mice have sex - specific elevated glucose uptake in brown adipose tissue ( bat ) . here we further explored the role of id2 in the regulation of core body temperature over the circadian cycle and the impact of id2 deficiency on genes involved in insulin signaling and adipogenesis in bat . we discovered a reduced core body temperature in id2/ mice . moreover , in id2/ bat , 30 genes including irs1 , ppars , and pgc-1s were identified as differentially expressed in a sex - specific pattern . these data provide valuable insights into the impact of id2 deficiency on energy homeostasis of mice in a sex - specific manner .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

PMC3603106

infectious diseases in type 2 diabetes can complicate diabetic ketoacidosis , derange hyperglycemia , or precipitate new onset diabetes . among infections , novel pandemic influenza virus h1n1 strain needs to be kept in mind when dealing with new onset diabetes with co - existing infectious agent , which , if present , is associated with high mortality . a 63-year - old female , with no known chronic illness , was hospitalized in month of aug 2010 with fatigue , breathlessness on exertion , productive cough with scanty sputum , and high - grade fever of 04 days duration . quick emergency assessment had shown temp 101f , tachycardia , tachypnea , p02 : 90% at room air , and normotensive . further evaluation revealed nho in both lung fields on chest x - ray , hyperglycemia 325 mg / dl , detected for first time . her symptoms and oxygen desaturation were out of proportion to clinical finding and chest x - ray findings . patient was managed with insulin infusion and empirical broad - spectrum antibiotic coverage in icu . as her condition worsened over next 12 hours , infection with novel influenza virus was thought and empirically started on oseltamivir after taking throat swab for h1n1 test and later , the sample was tested positive for h1n1 influenza by rt - pcr [ figures 1 and 2 ] . clinical course in the hospital was complicated by oxygen dependence requiring 10 - 12 ltr / hour of oxygen administration by nasal mask . oseltamivir was continued for 02 weeks with escalation of antibiotic coverage for any methicillin - resistant staphylococcus aureus organisms . appropriate prophylaxis against h1n1 was given to all nurses and paramedical staff and proper disposal of waste as per who guidelines . she made an uneventful recovery and was discharged on biphasic insulin @ 35 units / day with well - controlled blood sugar levels . chest x - ray showing pluffy opacities in both lung fields computed tomography scan chest : done at 48 hrs of admission , showing confluence of opacities with consolidation the first pandemic of 21 century , witnessed with detection of the first case in mexico 2009 , became evident that human - to - human transmission is caused by novel influenza virus , which is a re - assortment of 4 different strains of influenza virus genes causing novel h1n1 2009 pandemic strain . though it behaved like seasonal flu virus , the morbidity and mortality were more with risk factors like diabetes , pregnancy , cardiovascular status , obesity ; chronic illness suffered more from this pandemic flu . presently , we are in post - pandemic phase , and there are sporadic cases reported with mortality in high - risk cases [ figure 3 ] . the non - specific symptoms like fever , running nose , sore throat , body aches in flu make it difficult to identify and isolate cases initially [ table 1 ] . who has listed diabetes as silent - epidemic , which is prevalent worldwide , taking toll of deaths in the age group of 35 - 65 years ; 1 out of 10 deaths are attributed to diabetes , a ratio that increases to 1 out of 4 in certain vulnerable populations . people with diabetes and influenza are 3 times more likely to die from complications than without diabetes . immunological research from the hospital clinico universitario de valladolid in spain and the university health network found high levels of a molecule called interleukin 17 in the blood of severe h1n1 patients and low levels in patients with the mild form of the disease . two cases from iran with precipitation of diabetic ketoacidosis and h1n1 infection had fatal outcome . in a known diabetic , after h1n1 infection , hospitalization is known to triple and quadruple risk of icu admission once hospitalized . our case was complicated by precipitation of new onset hyperglycemia and occurrence of pneumonia with rapid decline in general condition . to best of our opinion , there were no reported cases of precipitation of diabetes with influenza a ( h1n1 ) pdm09 virus with positive outcome from india . [ source : who influenza laboratory surveillance information by the global influenza surveillance and response system ( gisrs ) . india , generated on 14/09/2012 06 : 14:47 utc ] distinguishing clinical features from common cold and flu this case lays emphasis on clinical acumen in recognition of a co - existing infectious process in a new onset diabetes mellitus , h1n1 infection , and prompt institution of oseltamivir to retard the progression of disease and decrease the mortality associated with it .

infectious diseases in type 2 diabetes can complicate diabetic ketoacidosis , derange hyperglycemia , or precipitate new onset diabetes . pulmonary tuberculosis being the most common . high index of clinical suspicion is required for co - existing h1n1 virus , which if present has high mortality if not treated . a 63-year - old female , with no known chronic illness , was hospitalized in month of aug 2010 with influenza - like symptoms and diabetes . quick evaluation revealed tachycardia , tachypnea , p02 90% at room air , and normotensive . clinical chest examination was normal . further evaluation revealed nho in both lung fields on chest x - ray , hyperglycemia 325 mg / dl , detected for first time . her signs and symptoms were out of proportion to clinical findings and chest x - ray findings . patient was managed with insulin infusion and empirical broad - spectrum antibiotic coverage in icu . as her condition worsened over next 12 hrs , infection with h1n1 was suspected and empirically started on oseltamavir after taking throat swab for h1n1 test and later , the sample was tested positive for h1n1 influenza by rt - pcr . clinical course in the hospital was complicated by oxygen dependence requiring 10 - 12 ltr / hr by nasal mask . she made an uneventful recovery . in a known diabetic , infection with h1n1 quadruples icu hospitalization , and only few cases of new onset diabetes with h1n1 were reported . two reported from iran had fatal outcome . this case emphasis on clinical acumen in recognition , and prompt institution of therapy will reduce associated mortality .

I

PMC3941966

the world health organization ( who ) estimated in a systemic review that glaucoma is the second commonest cause of blindness worldwide . in spite of this data , glaucoma was not included in the top priority five diseases for the first phase of the vision 2020 program of africa . this could be partly explained by the uncertainties that have existed concerning the evidence for case detection . with the scarcity of population - based surveys , many studies have assessed the clinical impact of glaucoma as regards visual fields and intraocular pressure as primary outcomes . this is optimum for developed countries but for developing countries , visual acuity as a primary outcome of assessment for glaucoma needs to be also studied . the aim of this clinic - based study is to assess patterns of visual acuity and blindness in glaucoma patients and to find out the bulk they represent in comparison to nonglaucoma patients in the same practice setting . patients attending cairo university hospital 's ophthalmic clinics were enrolled in this cross - sectional clinic - based study . inclusion criteria included all patients attending the ophthalmic outpatient clinic for one day of work , only one clinic for each unit , to avoid detection bias ( ophthalmology departments consist of subunits with each unit running separate clinics ) . tenets of helsinki declaration and the world medical association declaration on ethical considerations regarding health databases were respected . clinical data collected for these patients included their demographic data , best obtained visual acuity , and whether or not they have had an established diagnosis of glaucoma . ophthalmologists who collected the data did not participate in data analysis nor their interpretation to avoid performance bias . in this study , legal blindness was defined as visual acuity worse than 3/60 in the better seeing eye irrespective of the status of the visual field . visual impairment was defined as visual acuity 3/60 or better but worse than 6/18 in the better seeing eye irrespective of the status of the visual field . a totally blind eye was defined as an eye with vision of hand motions or less . legal blindness and visual impairment the definitions of legal blindness and visual impairment were guided by the egyptian law for the measurement of visual disability , which is exclusively dependent on the visual acuity to identify the degree of disability . the primary outcome from this study was to identify the levels of visual acuity affection in glaucoma patients in comparison to visual acuity of those without glaucoma in the same practice setting . the secondary outcome was to know the prevalence of glaucoma patients in this practice setting . nine hundred and eighty - eight eyes of 494 patients who had the mean age of 44.02 ( sd 18.8 ) years were enrolled in this survey . female to male ratio was 1.2 : 1 and their mean visual acuity was similar in either eye , 6/24 ( 3 snellen lines ) . the prevalence of patients with glaucoma was 8.1% ( confidence ; level 95% , interval 2.14.1 ) . the differences between glaucoma and nonglaucoma groups as regards age and sex were not statistically significant . patterns of visual acuities for glaucoma patients and nonglaucoma ones are shown in figure 1 . the weights of visual impairment and legal blindness when assessed per patient between either groups were not statistically significant ( p = 0.074 ) ( figure 2 ) . when analyzing eyes with total blindness , glaucoma ones had shown high odds of getting total blind eyes and that was statistically significant ( table 1 ) . one percent of africans are blind in whom glaucoma is the leading cause of irreversible blindness [ 5 , 6 ] . the earliest population - based survey for the patterns of visual acuity and blindness in egypt was done between 1965 and 1968 . they reported that the prevalence of patients with vision 1/60 or less were 16.7% higher in rural areas when compared to the urban areas , but they did not report the rates of legal blindness nor total blind eyes from glaucoma . however , we can find in their published tables that the prevalence of glaucoma was 2% in urban population and 9% in the rural one . few regional clinic - based reports have evaluated blindness of glaucoma in egypt . in these surveys from different egyptian cities , the prevalence of blindness from glaucoma was 12.1% at mansoura in 2002 , 19.7% at alexandria in 1987 , and 7.6% at al - azhar university study ( cairo ) in 1989 [ 810 ] . similarly , the proportion of blindness from glaucoma in clinic - based surveys from different african countries ranged from 10% to 33% . in a hospital - based study in nigeria , researchers reported that 23.1% of blind patients were due to glaucoma . we found highly significant odds for recording eyes with total blindness in eyes with glaucoma ( 30% ) . in a hospital - based study in addis ababa , this could be either due to a delay in presentation , diagnosis , or many other factors . the definitions of blindness at which visual acuities were assessed were not uniform among the previous studies . in the current report which was done in cairo , the better figures of legal blindness rates compared to earlier studies , either reflects improvements in glaucoma health care or was underestimated because we did not include visual fields in our legal blindness assessment , which would have increased those who were legally blind . amending the national laws and considering the status of visual fields when assessing the visual disability is recommended to fulfill the requirements advised by the who regarding legal blindness and visual impairment . the high rates of the totally blind eyes found for glaucoma in this study mandates the importance to investigate the factors behind these figures on a wider scale . this necessitates the need for interventional strategies to manage the problem of glaucoma among egyptian patients .

purpose . glaucoma is the second commonest cause of blindness worldwide . visual fields and intraocular pressures are optimum outcomes to be assessed in developed countries . visual acuity as an outcome is the key player when assessing blindness in developing countries . the aim of this study is to assess visual impairment and blindness in glaucoma patients and to identify the bulk they represent in comparison to nonglaucoma patients in the same practice setting . methods . patients attending outpatient clinics of cairo university hospitals were enrolled in this cross - sectional study . clinical data collected for these patients included their demographic data , best obtained visual acuity , and whether or not they have had an established diagnosis of glaucoma . results were compared at 95% confidence intervals . results . 988 eyes of 494 patients were included for this study . their mean visual acuity was 6/24 ( 3 snellen lines ) . legal blindness was found in 5% and 4% of glaucoma and nonglaucoma groups , respectively . there were high odds for finding eyes with total blindness due to glaucoma . that was statistically significant ( p < 0.05 ) . conclusions . a large proportion of glaucoma patients suffered from blindness in at least one eye . interventional strategies are recommended regarding visual disability among egyptian glaucoma patients .

1. Introduction 2. Methods 3. Results 4. Discussion

PMC4563293

neck pain and its disability result in a significant burden on individuals and family members , communities , health - care systems , and businesses1,2,3 . recently , studies have been conducted in an attempt to identify the disability of deep cervical muscles contributing to stabilization of the cervical spine . a comparison study reported that people with neck pain have reduced activity of deep neck flexor ( dcf ) muscles with excessive activity of long - lever superficial muscles ( e.g. , sternocleidomastoid and anterior scalene muscles ) , resulting in unstable segmental motion in the intervertebral joint during repetitive upper limbs tasks4 . disability of the dcf muscles in people with neck pain is regarded as a general consideration that a health professional might consider in planning a treatment strategy . in a control trial , jull et al.5 reported that targeted training of the dcf muscles in people with neck pain demonstrated a greater reduction in score for the neck disability index ( ndi ) and intensity of neck pain . however , for health professionals , the only feasible assessment method known to detect the activity of the dcf muscles in a clinical setting , due to their location , is electromyographic testing by applying needle or tube insertions , which is not a simplistic approach clinically and legally . therefore , in a clinical setting , an easy to apply approach may benefit rehabilitation practices for the imbalance of muscle recruitment in neck flexor muscles due to lack of dcf muscle activity . ultrasonography is currently receiving attention as a tool for examination of deep layer muscles , but few studies have successfully reported muscle recruitment and neuromuscular responses of dcf muscles using ultrasonography6 . the purpose of this study was to identify the imbalance of muscle recruitment in cervical flexor muscles during the craniocervical flexion test ( ccft ) using ultrasonography in people with neck pain and to use the findings to further guide explicit exercise management for neck flexor disabilities . the study subject were 18 students ( 9 males ) from d university gyeongbuk province with a mean age , weight , and height of 21.03 2.67 years , 57.62 12.9 kg , and 168.01 7.61 cm , respectively . participants were invited to participate in the study if they reported a current neck pain score of 5 points or greater for the ndi ( total score is 50 points)7 . participants were excluded if they reported signs of neurological disorders , visited a health professional , or had experienced whiplash . in accordance with the ethical standards of the declaration of helsinki , information on the study was provided to all the subjects , and informed consent was received from them prior to participation in the study . real - time ultrasonography images were obtained from the longitudinal cross section of the right deep cervical flexor unit ( longus colli and capitis ) and right sternocleidomastoid during the ccft in accordance with the previously established protocol6 . a 7.5 mhz transducer of a z.one ultra convertible ultrasound system ( zonare medical systems , inc . , mountain view , ca , usa ) was positioned diagonally against the front of the neck area running along the trachea s line and 5 cm away from the midline of the cervical spine . subjects were positioned in the supine position with the head and neck in a mid position , and the task required subjects to perform five gradually increasing movements of craniocervical flexion motion adjusted by visual feedback from an air - filled pressure sensor right underneath their neck ; the applied pressures were 22 , 24 , 26 , 28 , and 30 mmhg the images obtained from the trial were transmitted to the nih imagej software ( version 1.44 for windows ) and subjected to image analysis process to measure the muscle thicknesses . a four - line grid with a 0.5 cm interval between lines was drawn on each image , and the first line was centered in the middle of each image . the average value for distance estimated from the second , third , and fourth line was designated as the thickness of each muscle . the amount of change in muscle thickness at each pressure level from the reference pressure ( 20 mmhg ) was calculated by the following formula : ( muscle thickness at the target pressure muscle thickness at the reference pressure ) / muscle thickness at the reference pressure8 . the imbalance of muscle recruitment at each pressure level was represented as the ratio of the change in muscle thickness of the sternocleidomastoid muscle ( scm ) to the change in muscle thickness in the dcf muscles ( imbalance ratio : thickness of scm / thickness of dcf ) . this study used ibm spss statistics for windows ( version 20.0 ) to analyze the data . repeated measures analysis of variance ( anova ) was used to examine the ratio of the amount of muscle thickness changes in two comparison muscles according to incremental cervical flexion movements . the muscle thickness ratio of the scm / dcf according to the incremental pressure showed significant differences between 22 mmhg and 24 mmhg , between 24 mmhg and 28 mmhg , between 24 mmhg and 30 mmhg , and between 26 mmhg and 28 mmhg ( p<0.05 ) . the muscle thickness ratio of the scm / dcf was smallest at 24 mmhg ( table 1table 1.comparison of the ratio of dcf thickness to scm thickness according to pressure increasepressure22 mmhg24 mmhg26 mmhg28 mmhg30 mmhg22 mmhg2.281.271.330.42 * 1.470.471.940.602.040.8124 mmhg1.470.471.940.60 * 2.040.81 * 26 mmhg1.940.60 * 2.040.8128 mmhg2.040.81*p<0.05 ( meansd ) . in this study , ultrasonographic assessment of the muscle recruitment in cervical flexor muscles was conducted in people with neck pain . the secondary aim of this study was to determine the clinical applicability of using ultrasonography in evaluating the disability of cervical flexor muscles . multiple studies have successfully visualized the recruitment pattern of deep cervical muscles in noninvasive and low - cost manners6 , 9,10,11 , and the present study highly suggested that ultrasonography could be used for this purpose . the real values for the thicknesses of the dcf and scm gradually increased as the pressure increased . heightened recruitment of the scm during the ccft , was observed in the present study , which is consistent with the results of previous studies5 , 12 , 13 . nonetheless , the aim of this study , unlike the previous studies , was to compare the muscle recruitment ratios of the superficial and deep cervical muscles and to suggest the optimal level of pressure for balanced muscle recruitment during the ccft . as depicted in table 1 , the greatest imbalance in muscle recruitment between the deep and superficial muscles during the ccft was revealed at a pressure of 22 mmhg due to considerable thickness changes in the scm . the second and third greatest imbalances , also due to increased changes in recruitment of the scm , were 30 mmhg and 28 mmhg , respectively , while the dcf muscles maintained steady amounts of recruitment changes at both of these pressure levels . this finding suggests that health professionals need to take into consideration differences in applied pressures during interventions for patients with a disability of the dcf . as the activation of the scm during the ccft is smallest at the pressures of 24 mmhg and 26 mmhg , a recommendation for an adequate exercise intervention for dcf training might be established at a pressure level between 24 mmhg and 26 mmhg . although this study found great imbalances at pressure levels of 22 mmhg , 28 mmhg , and 30 mmhg , the standard muscle recruitment ratio between deep and superficial cervical muscles was not evaluated in a healthy population . for a comprehensive assessment of the muscle recruitment between symptomatic and non - symptomatic populations ,

[ purpose ] the purpose of this study was to identify the imbalance of muscle recruitment in cervical flexor muscles during the craniocervical flexion test by using ultrasonography and to propose the optimal level of pressure in clinical craniocervical flexion exercise for people with neck pain . [ subjects and methods ] a total of 18 students ( 9 males and 9 females ) with neck pain at d university in gyeongsangbuk - do , south korea , participated in this study . the change in muscle thickness in superficial and deep cervical flexor muscles during the craniocervical flexion test was measured using ultrasonography . the ratio of muscle thickness changes between superficial and deep muscles during the test were obtained to interpret the imbalance of muscle recruitment in cervical flexor muscles . [ results ] the muscle thickness ratio of the sternocleidomastoid muscle / deep cervical flexor muscles according to the incremental pressure showed significant differences between 22 mmhg and 24 mmhg , between 24 mmhg and 28 mmhg , between 24 mmhg and 30 mmhg , and between 26 mmhg and 28 mmhg . [ conclusion ] ultrasonography can be applied for examination of cervical flexor muscles in clinical environment , and practical suggestion for intervention exercise of craniocervical flexors can be expected on the pressure level between 24 mmhg and 26 mmhg enabling the smallest activation of the sternocleidomastoid muscle .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

PMC4045310

dietary supplement intake , in addition to training , appropriate nutrition , and advantageous gene polymorphisms , is recognized to confer exercise performance benefits [ 14 ] . such supplements ( e.g. , caffeine , bicarbonate , creatine , and beta alanine ) are widely consumed by athletes in many sports . the role of caffeine as an ergogenic aid the study by costill et al . was the first to demonstrate improved time - to - exhaustion ( tte ) exercise with acute preexercise ingestion of caffeine . subsequent investigations reported that caffeine intake can improve cycling [ 6 , 7 ] , high intensity running [ 8 , 9 ] , and repeated sprint running performance . a recent review on the effects of caffeine on exercise performance suggested that , at least in endurance sports , even small to moderate caffeine doses ( 2 - 3 mg / kg of body mass ) are likely to confer performance benefits . caffeine induces numerous physiological effects . although its stimulating effects on exercise performance in humans were initially thought to occur via increased fatty acid oxidation rates and muscle glycogen - sparing effect [ 5 , 7 ] , several studies reported conflicting findings [ 12 , 13 ] . in addition to peripheral actions , caffeine was shown to reduce perception of effort during exercise , suggesting that some ergogenic effects may be partially accounted for by caffeine - mediated maintenance of central nervous system ( cns ) drive . specifically , meeusen et al . implied that the performance - enhancing effects of caffeine are likely to be linked with the antagonism of adenosine receptor signalling in the brain . beetroot juice consumption has recently drawn attention of many endurance athletes as evidence accumulates indicating its performance - boosting effects . several independent research groups have demonstrated in double - blind , placebo - controlled studies that acute as well as chronic intake of beetroot juice containing about 8 mmoles of nitrate ( no3 ) can improve endurance and high - intensity intermittent performance [ 1620 ] . following absorption , nitrate reappears , via enterosalivary circulation , in the mouth , where it is degraded to nitrite ( no2 ) by facultative anaerobic bacteria on the surface of the tongue . further nitrite conversion to nitric oxide ( no ) takes place in stomach . although the exact mechanisms by which increased beetroot juice - derived no2 and no bioavailability enhance exercise performance remain to be fully elucidated , improved muscle mitochondrial function and/or reduced high - energy phosphate turnover leading to a decreased oxygen cost of exercise are likely to be involved [ 16 , 19 ] . considering that acute preexercise ingestion of caffeine and nitrate - rich beetroot juice has been found to enhance exercise performance via different mechanisms ; their combined effects on performance could potentially be additive . in other words the preexercise ingestion of beetroot juice and caffeine could improve performance more than intake of either beetroot juice or caffeine alone . there are relatively few studies on the effects of combining different dietary ergogenic aids , and to our knowledge , no study has investigated the potential combined ergogenic effects of nitrate and caffeine . therefore , the primary aim of this randomised , double - blind , placebo - controlled study was to evaluate potential additive effects of acute preexercise beetroot juice and caffeine supplementation on time to exhaustion during cycling at 80% vo2max in healthy , well - trained male participants . a secondary aim was determine if nitrate 's effects on the oxygen cost of exercise are influenced by caffeine co - ingestion . we hypothesised that the combination of ingesting both beetroot juice and caffeine before exercise would have greater performance enhancing effects than beetroot juice or caffeine alone . fourteen well - trained male participants volunteered to participate in this randomized , double - blind , placebo - controlled design study . their age , height , body mass , and vo2max were 22 3 years , 177 1 cm , 76 7 kg , and 63 10 ml / kg / min , respectively ( values are expressed as mean sd ) . all participants engaged in endurance sports at least 3 times per week and all were familiar with cycling . all participants were fully informed of experimental procedures , associated risks , and a rationale of the study before they gave their written consent to participate in this study . participants were included if they were nonsmokers , currently healthy , had been training for at least 2 h per week of moderate to vigorous physical activity , and had no known history of cardiac , hepatic , renal , pulmonary neurological , gastrointestinal , haematological , or psychiatric illness . all exercise tests were performed under controlled environmental conditions ( 22 1c , 5060% relative humidity ) . approximately one week before supplementation trials participants visited the laboratory to undertake screening , vo2max determination , and familiarization tests . upon arrival , participants ' weight was measured to the nearest 0.1 kg using a digital scale ( seca 770 , seca ltd , germany ) . each subject performed a continuous incremental exercise test on one of the two electromagnetically braked cycle ergometers ( lode excalibur sport , groningen , the netherlands and lode corvial , groningen , the netherlands ) to volitional exhaustion to determine vo2max . subjects were randomly assigned to either one of the two cycle ergometers and the allocation was maintained during the supplementation trials . a fan was set approximately 2 m away from participants to provide cooling during exercise and supplementation trials . self - selected saddle and handlebar settings used were replicated for all subsequent supplementation trials . participants began cycling at 95 w , with increments of 35 w every 3 min until volitional fatigue despite strong verbal encouragement . samples of expired gas were collected in douglas bags during the third minute of each work rate increment , and heart rates were measured continuously using short - range telemetry ( polar , kempele , finland ) . a paramagnetic oxygen analyser ( servomex 1420b , crowborough , uk ) and an infrared carbon dioxide analyser ( servomex 1415b ) were used to determine percentages of expired oxygen and carbon dioxide . these were used together with a dry gas meter ( harvard apparatus , edenbrige , uk ) for a determination of rates of oxygen consumption ( vo2 ) and carbon dioxide production ( vco2 ) . maximal oxygen consumption was determined from the expired gas sample collected during the final minute of the exercise test when subjects indicated that they could only continue exercise for an additional minute . work rates corresponding to 60% and 80% vo2max were interpolated from the vo2-work rate relationship . after 30 min recovery participants completed a 30 min familiarization trial at 60% vo2max , which followed the same format as the experimental trials . this was undertaken to ensure that the subjects were accustomed to the procedures used during the investigation and to minimise any potential learning or anxiety effects . following completion of the preliminary tests each participant visited the laboratory on four occasions interspersed by at least four days to complete 30 min exercise at 60% vo2max immediately followed by a time - to - exhaustion trial at 80% vo2max . the treatments on these trials were as follows : ( 1 ) placebo ( pla ) , ( 2 ) placebo + caffeine ( pla+c ) , ( 3 ) beetroot juice ( br ) , and ( 4 ) beetroot juice + caffeine ( br+c ) . the treatments were administered using a randomized double - blind , placebo - controlled study design . a br+pla trial was not included because that would have supplied only half of the required amount ( 8 mmoles ) of nitrate . on test days , participants arrived at the laboratory after an overnight fast in a rested state avoiding strenuous physical activity the day before at either 09:00 or 11:30 . after ~10 min rest they consumed a standardized breakfast containing 127 kcal ( 24 g of carbohydrate , 2.8 g of fat , and 1.6 g of protein ) and a 70 ml concentrated beetroot juice ( beet - it , james white drinks ltd , ashbocking , uk ) containing 4 mmol no3 or placebo ( nitrate - depleted beet - it containing < 0.1 mmol no3 from the same manufacturer ) . after 75 min participants consumed another 70 ml concentrated beetroot juice or placebo with or without caffeine ( 0.5 mg / kg body mass ) . following another 75 min rest period subjects performed 30 min cycling at 60% vo2max followed by a time - to - exhaustion ( defined as inability to maintain pedalling cadence ) trial at 80% vo2max . during supplementation trials water the timing and dosages of caffeine and nitrate were based on previous studies that have demonstrated ergogenic effects in endurance exercise with 5 mg / kg caffeine provided 1 - 2 hours before exercise [ 46 , 9 , 11 , 13 , 14 ] and 8 mmoles of dietary nitrate provided 2 - 3 hours before exercise [ 1619 , 2224 ] . expired gas samples data were collected for one minute during the tenth and twentieth minute of the initial 30 min exercise and during the seventh minute of the 80% vo2max time - to - exhaustion exercise along with participants ' heart rate and rating of perceived exertion ( rpe ) collected every 5 min using the borg 620 scale . participants were unaware of the cycling duration during the trials and were given constant encouragement to cycle to exhaustion . rates of whole - body fat and carbohydrate oxidation were calculated from measured gaseous exchange variables as previously described . to help ensure that metabolic conditions were similar prior to each experimental trial , participants were asked to record dietary and fluid intake during the day before the first trial and replicate it during the day before the subsequent supplementation trials . excessive caffeine ( > 5 cups of tea , coffee , and coke per day ) and nitrate - rich foods ( e.g. spinach , rocket , lettuce , and radish ) were not permitted over the course of the study . saliva samples , for salivary cortisol , nitrite , and nitrate determination , were collected before supplement ingestion , prior to exercise , during the last 5 min of the ride at 60% vo2max , and 10 min after completion of the time - to - exhaustion test . water intake was not permitted 5 min prior to saliva collection at any time point to avoid salivary analyte dilution . briefly , participants were seated and provided an unstimulated saliva sample by passive dribble into a 7 ml polypropylene collection tube ( sterilin , caerphily , uk ) over a course of 24 min with opened eyes , slightly bent forward with minimal orofacial movement . all samples were immediately centrifuged at 12 000 rpm for 2 min and the supernatant was collected and stored at 20c for further analysis . the salivary cortisol concentrations were determined in duplicate using an elisa kit ( salimetrics , state college , pa , usa ) . duplicate salivary nitrate and nitrite were analysed using a colorimetric assay kit ( cayman chemicals , usa ) . the intra - assay coefficient of variation for cortisol , nitrate , and nitrite was < 3.6% . rpe , physiological , and salivary cortisol variables were analysed using repeated measures anova or friedman anova for normally and nonnormally distributed data , respectively . post hoc sidak or wilcoxon signed rank test was used to detect significant differences between groups where these were apparent for normally and nonnormally distributed data , respectively . data in the text and tables are presented as mean and standard deviation ( sd ) . all statistical analyses were carried out using spss 19.0 for windows ( spss , chicago , il , usa ) . friedman 's anova revealed lower rpe during tte exercise at 15 min of 80% vo2max on br+c compared with pla and pla+c ( p = 0.016 and p = 0.047 , respectively ) as shown in table 1 . there was a tendency for rpe to be reduced at 10 and 15 min of 80% vo2max on br+c compared with pla ( p = 0.081 ) and br ( p = 0.084 ) , respectively . there was no interaction effect for hr during 30 min exercise at 60% vo2max and 80% vo2max and rpe during 30 min 60% vo2max exercise between the trials ( data not shown ; p > 0.05 ) . no significant differences ( p 0.104 ) between treatments were found in any other measured variable as shown in tables 2 and 3 . resting preexercise salivary nitrite and nitrate concentrations were higher as shown by the paired t - test and wilcoxon signed rank test after beetroot juice ingestion ( p 0.002 ) than before ( figures 1(a ) and 1(b ) ) . on the contrary , no differences in salivary nitrate and nitrite were found between preingestion and preexercise samples in pla and pla+c ( p > 0.05 ) . no significant differences in time - to - exhaustion protocol ( f(3,39 ) = 2.269 , p 0.096 ) between trials were found ( figure 2 ) ; however , exercise time in br + c tended ( p = 0.096 ) to be higher than in pla ( 1463 774 versus 1003 480 s ) , corresponding to a 46% improvement . similarly , 18% and 27% nonsignificant time - to - exhaustion improvements were observed on br+c trial compared with br and pla+c alone , respectively . the individual performance data demonstrate that eleven , nine , and eight subjects of fourteen cycled longer on br+c compared with pla , br , and pla+c , respectively ( figure 3 ) . there was a main effect of time with salivary cortisol being higher during exercise compared to preexercise ( p < 0.05 ) . likewise , salivary cortisol was found to be higher postexercise compared to preexercise and during exercise ( p < 0.05 ) with no differences between supplementation trials ( p > 0.05 ) . the main purpose of the present study was to evaluate potential additive effects of beetroot juice and caffeine co - ingestion on tte exercise performance . the other main goal of the experiment was to test the hypothesis if dietary nitrate reducing effects on oxygen uptake are influenced by caffeine ingestion . the major finding of this study was an 18% and 27% nonsignificant improvement in tte at 80% vo2max on br+c compared to br and pla+c , respectively . this finding supports our hypothesis implying potential positive and additive effects of br+c on exercise capacity in well - trained male athletes . to our knowledge , this is the first study demonstrating potential beneficial effects of combined acute preexercise intake of beetroot juice and caffeine on tte performance . our findings show that acute preexercise co - ingestion of beetroot juice and caffeine results in lower rpe , providing a potential mechanism , by which br+c may improve exercise performance . reduced rpe in br+c compared to pla and pla+c suggests that beetroot and caffeine co - ingestion postponed the development of exercise intolerance and/or reduced perception of effort during exercise . as this finding was not observed for the pla+c treatment , it is plausible that the reduction in rpe represents a br+c - specific effect . the physiological importance of this finding is supported by the doherty and smith meta - analysis indicating that caffeine - mediated improved exercise capacity can be largely explained by reduced rpe . preexercise caffeine intake alone has been suggested to activate neuroendocrine responses ( e.g. , noradrenaline secretion ) to promote lipolysis and a glycogen - sparing effect [ 7 , 10 ] . results from studies using femoral arterial - venous catheters and muscle biopsy samples before and after exercise have called this caffeine - mediated glycogen - sparing effect into question [ 26 , 27 ] . the findings of our study support this view . we did not observe any difference in salivary cortisol between trials , nor did we find any difference in whole - body carbohydrate or fat oxidation rates . these findings imply that acute preexercise caffeine intake alone or in combination with beetroot juice does not affect neuroendocrine responses and whole - body metabolic substrate utilisation during exercise in humans . alternatively , a recent study indicated that caffeine may improve interstitial potassium handling during exercise , which is suggested to play a crucial role in fatigue development [ 10 , 28 ] . therefore , it can be argued that caffeine may have also acted via another peripheral mechanism that we did not measure . the findings of the present study further support the ergogenic effects of beetroot juice intake on exercise performance in humans . for instance , in a randomized , cross - over design study , acute consumption of beetroot juice resulted in improved 4 km and 16 km time trial performance in club - level competitive cyclists . others have found that acute and chronic beetroot juice intake improves high - intensity tte performance , while reducing oxygen uptake [ 22 , 23 ] . collectively , these findings suggest that both acute and chronic beetroot juice intake improve capacity to perform exercise in humans . beetroot juice - derived nitrate , nitrite , and no are thought to act via multiple mechanisms . dietary nitrate supplementation was shown to reduce the oxygen cost of exercise during both low- and moderate - intensity exercise in humans . using p magnetic resonance spectroscopy ( mrs ) chronic dietary nitrate supplementation attenuated the reduction in high - energy phosphate concentration ( phosphocreatine ) and reduced atp turnover in humans during low- and high - intensity knee - extensor exercise , suggesting that the lower oxygen cost of exercise may be , at least partially , explained by a reduced atp cost of muscle contraction . in another study , larsen et al . three days of sodium nitrate intake resulted in increased maximal atp production by isolated mitochondria and reduced mitochondrial oxygen affinity . interestingly , during submaximal exercise , the nitrate supplementation treatments displayed lower oxygen consumption and higher rer suggesting more efficient energy utilisation . in mice , 7 days of sodium nitrate supplementation was shown to increase intact muscle fibre force production via greater intramuscular calcium concentration , suggesting another potential mechanism by which muscle performance may be enhanced . taken together , these findings suggest that increased nitrate bioavailability is involved in the regulation of intramuscular calcium handling , high - energy phosphate utilization , and oxidative metabolism . more recently , peacock et al . reported that acute preexercise potassium nitrate consumption resulted in no improvement in 5 km time - trial performance in elite cross - country skiers . further , cermak et al . found that consumption of a single dose of concentrated beetroot juice did not affect 1 h time - trial cycling exercise performance . in a similar fashion , a 3-day period of sodium nitrate supplementation was found to cause no improvement in distance covered or mean power output over a 40 min time - trial . although these studies imply that acute and/or chronic beetroot juice intake may not confer an exercise performance benefit , a recent review by hoon et al . indicates that , even if not reaching statistical significance , a small positive effect on time trial and graded exercise performance may be meaningful in an elite sport context . the other main finding of the present study was that the oxygen cost of exercise at 60% and 80% vo2max on br+c was unaffected by caffeine compared with beetroot juice alone , as might have been assumed if more free fatty acids were being oxidized following caffeine consumption ( i.e. , oxidation of free fatty acids requires ~10% more oxygen than carbohydrate to produce the same amount of energy ) . this finding demonstrates that caffeine alone or in combination with beetroot juice does not affect muscle substrate utilisation and oxygen consumption during exercise . several potential factors may partially explain these discrepancies , including the exercise protocols used , participants ' fitness levels , and the form of nitrate provided . although the overall magnitude of exercise improvement on br+c may suggest physiological significance , no statistical difference between treatments was detected . this discrepancy may be , in part , explained by the study design , and thus statistical methods used to test for differences , and high interindividual variability in the tte protocol . the individual performance data demonstrated that eleven out of fourteen and nine out of fourteen participants improved their exercise capacities on br+c versus pla and br , respectively , suggesting a consistent general trend for performance enhancement on br+c , yet a range of 2613 sec was observed between the longest and the shortest br+c trial . this demonstrates that despite a general tendency for performance improvement , highly variable responsiveness exists between individual participants . this high interindividual variability may be , at least , partially explained by tte protocol , in which participants were shown to display high test - retest variability . for instance , up to 27% versus < 4% day - to - day variability has been reported for tte and time - trial protocols , respectively , suggesting that further research is warranted to fully clarify effects of combined beetroot and caffeine intake on human exercise performance . in summary , acute preexercise beetroot juice co - ingestion with caffeine augmented time - to - exhaustion at 80% vo2max performance by 18% and 27% over beetroot and caffeine alone , respectively . moreover , 46% enhancement in exercise capacity was observed in br+c compared with placebo , likely attributable to lower rpe in br+c trial . there appears to be a strong likelihood that the ergogenic effects of dietary nitrate and caffeine are additive . therefore , endurance athletes may benefit from this preexercise acute nutritional strategy prior to participation in competitive endurance events .

aims . to evaluate the possible additive effects of beetroot juice plus caffeine on exercise performance . methods . in a randomized , double - blinded study design , fourteen healthy well - trained men aged 22 3 years performed four trials on different occasions following preexercise ingestion of placebo ( pla ) , pla plus 5 mg / kg caffeine ( pla+c ) , beetroot juice providing 8 mmol of nitrate ( br ) , and beetroot juice plus caffeine ( br+c ) . participants cycled at 60% maximal oxygen uptake ( vo2max ) for 30 min followed by a time to exhaustion ( tte ) trial at 80% vo2max . saliva was collected before supplement ingestion , before exercise , and after the tte trial for salivary nitrate , nitrite , and cortisol analysis . results . in beetroot trials , saliva nitrate and nitrite increased > 10-fold before exercise compared with preingestion ( p 0.002 ) . tte in br+c was 46% higher than in pla ( p = 0.096 ) and 18% and 27% nonsignificant tte improvements were observed on br+c compared with br and pla+c alone , respectively . lower ratings of perceived exertion during tte were found during 80% vo2max on br+c compared with pla and pla+c ( p < 0.05 for both ) . conclusions . acute preexercise beetroot juice coingestion with caffeine likely has additive effects on exercise performance compared with either beetroot or caffeine alone .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions

PMC3043451

unfortunately , it is fraught with high complication rates , extended hospital stays , and an unacceptably high recurrence rate that is underestimated by the very surgeons performing the operation . since its introduction to the surgical community 8 years ago , the process of repairing ventral / incisional hernias by laparoscopic techniques has consistently demonstrated a remarkable reduction in recurrence rates , hospital stays , and morbidity . the purpose of this paper is to introduce a technique for laparoscopic repair of ventral / incisional hernias ( livh ) that is less complex and time consuming than previously reported procedures . like our predecessors , we rely on a wide overlap of a synthetic material intraabdominally to cover the hernia . we have chosen to use composix mesh and composix e / x mesh ( davol inc . , cranston , ri ) for these repairs because the materials utilized in the construction of this mesh allow it to be secured rapidly without the need for transfixion sutures through the abdominal wall . six years of operative experience repairing a wide variety of hernias with this technique has confirmed our position that it is an easily learned technique . more importantly , it grants all the benefits of laparoscopic surgery to patients with ventral / incisional hernias while maintaining recurrence rates that are superior to recurrence rates of open repairs . three steps are required that must be accomplished if the ventral / incisional hernia is to be repaired in a satisfactory fashion : ( 1 ) safe abdominal access with proper port geometry , ( 2 ) complete lysis of intraabdominal adhesions , and ( 3 ) introduction and fixation of an appropriately sized piece of mesh . in many ways , they are made to understand that before any mesh is inserted the adhesions must be removed so that anatomy is clearly seen . a full bowel prep ( cathartics and antibiotics ) is given the evening before surgery to improve the safety of intraoperative bowel handling and adhesiolysis . we also describe the need for a compressive dressing over the hernia sac in the postoperative period . a 50% reduction in postoperative seromas has been demonstrated with the use of postoperative abdominal wall pressure dressings . in most cases , our patients are asked to continue the compressive dressing until the first postoperative visit 7 to 10 days postoperatively . the procedure is performed with the patient under general anesthesia , and intravenous antibiotics are given in the holding area . in most cases , 1.5 g of ampicillin / sulbactam is given unless patient allergies dictate a change . an orogastric tube is inserted , but a foley catheter is placed only at the discretion of the surgeon . the patient is placed in a low lithotomy position with both arms outstretched on arm boards . typically , a towel roll is placed under the patient 's flank on the primary surgeon 's side . this rolls the patient and target hernia slightly away from the surgeon , which facilitates placement and utilization of the trocars in the lateral abdominal wall . all lines and tubing leave the field at the patient 's shoulders so that the surgeon may move freely from the patient 's left to right sides or even between the legs if safe dissection requires it . initial port strategy is determined by the location of the hernia but may be altered by the degree of adhesiolysis required . in most cases , typically , a single 10- to 12-mm trocar is inserted in a left subcostal position , and 5-mm ports are placed at the transverse umbilical line and just anterior to the anterior superior iliac spine . in all cases , the method of initial port - site insertion is left to the surgeon 's preference and experience . we prefer nonbladed optical ports ( optivew , ethicon endo surgery , cincinnati , oh ) inserted in an oblique or z - track fashion . our personal experience has shown that when properly inserted they are safe and reduce port - site hernias and complications . often a fourth 5-mm port placed 180 degrees from the previous ports is all that is required . the goals of port placement are to maintain proper triangulation for working , and to eliminate camera angles that result in paradoxical motions on the screen . we feel that the ports need to be far enough away from the hernia so that angled scopes are not required to visualize the defect . this strategy allows us to gain maximum utility from our nonreticulating 5-mm instruments and tacker . over - reliance on angled laparoscopes during a case would suggest that the ports have been inserted too close to the hernia defect . when epigastric , suprapubic , and flank / lumbar hernias are encountered , the port strategy and patient positioning are adjusted . in these special circumstances , we take full advantage of table tilt , patient positioning , and flexible port placement to accomplish the same port strategy goals mentioned above . suprapubic hernias often require a limited preperitoneal dissection to push the bladder down and expose the pubic tubercle and cooper 's ligament for mesh fixation . epigastric hernias typically require a complete takedown of the falciform ligament so that the mesh may extend under the costal margin for added security . we avoid port placement or mesh insertion though the hernia defect itself as any wound complication with such a port could potentially affect or expose the mesh below it . once the ports are appropriately placed , the abdominal cavity is evaluated and any required adhesiolysis is accomplished . all hernia contents are reduced , but no effort to remove the sac is undertaken . if the hernia sac contains bowel , the contents are evaluated to ensure that bowel obstructions will not occur from intraloop adhesions now that they have been mobilized and reduced . blunt dissection is very useful but well vascularized and dense intraabdominal adhesions are lysed with the curved 5-mm lcs harmonic scalpel ( ethicon endo surgery , cincinnati , oh ) . its multi - tasking nature ( grasping , dissection , transection , and hemostasis ) along with its safety profile relative to monopolar cautery has made it indispensable for many of these cases . when dense intestinal adhesions to prior incisions or mesh make utilization of any energy source hazardous , gentle blunt and sharp dissection with laparoscopic scissors and graspers is used . once the edges of the hernia are clearly visible , the defect size is measured internally by insertion of a small flexible plastic ruler or umbilical tape . external measurements overestimate the true size of the fascial defect when the abdomen is insufflated and are likely to promote insertion of a mesh that is far larger than needed . our goal is to place a prosthesis that extends 3 to 5 cm beyond all edges of the defect . five cm is the preferred overlap but in some circumstances , such as fixation of the mesh to the pubic tubercle or under the costal margin , smaller overlaps are permitted . we have made a transition from using the original composix mesh to the new composix e / x mesh for our repairs . we have found the expanded polytetrafluoroethylene ( eptfe ) surface on the composix e / x to be more resistant to abrasion during manipulation . the mesh is also thinner than its predecessor , which has advantages during mesh insertion . once the proper size has been determined , it is inserted via the 10- to 12-mm trocar site . we have found that 18 x 23-cm pieces of mesh can be wound around a 5-mm grasper and inserted through the trocar itself . it is important to roll the mesh so that the eptfe surface is on the inside and protected from abrasions from the valves in the trocar . larger pieces of mesh up to and including the 25 x 36-cm piece may be rolled and inserted via the port site after the port is removed . direct insertion via the abdominal wall trocar incision requires the surgeon to roll the mesh so that the marlex surface is on the inside . this prevents unnecessary drag / trauma as the mesh is inserted through the layers of the abdominal wall . the unique incorporation of marlex on the peritoneal side of this particular mesh has allowed us to eliminate the full thickness abdominal wall sutures required for placement and proper fixation of eptfe patches . by eliminating this step , transabdominal transfixion sutures are not necessary for securing or orienting the mesh . the marlex backing on the mesh results in excellent handling characteristics , which simplifies orientation and accurate placement . in most cases , it can be held in place against the abdominal wall with a single 5-mm instrument prior to tack placement . it is recommended that the midline of the mesh be oriented with the vertical axis of the hernia prior to placing the tacks . the first tack should be placed at either the caudal or cephalad edge of this vertical axis . the second tack is then placed 180 degrees from the first after a final check of mesh orientation . drawing a dark line down the vertical axis of the mesh with a sterile marking pen prior to mesh insertion is recommended . when done properly , this technique eliminates the potential for inadequate overlap of the mesh beyond the edges of the hernia . once the mesh is properly positioned with the first 2 tacks , the mesh is rapidly secured with a row of tacks at the periphery of the mesh . these are placed 1 to 2 cm apart to transfix the mesh and to prevent bowel or omentum from slipping under the edge . a second inner row of tacks is placed at the edge of the hernia defect itself with bimanual palpation ( hand outside / tacker inside ) to ensure proper placement . one hundred consecutive patients have undergone laparoscopic repair of their ventral and incisional hernias with the techniques described above over a 27-month period . most were elective surgeries ; however , 4 cases of incarceration and obstruction were included . three enterotomies ( 2 small bowel , 1 right colon ) occurred in the elective surgical group during blunt or sharp dissection . in each case , no contamination occurred , the bowel was repaired laparoscopically in 2 layers , and mesh was inserted without complication . no injuries occurred related to the use of the harmonic scalpel or the nonbladed optiview trocars . the complexity of the cases has varied widely as has the size of the defects repaired . mesh inserted for repair has varied in size from 10 x 15 cm to 25 x 36 cm . cranston , ri ) fused to 1 layer of eptfe was used for the repairs . since the introduction of composix e / x , we no longer use the original mesh . the current mesh has 1 layer of marlex sewn to a thicker layer of eptfe . consequently , the eptfe surface is more resistant to abrasion during placement , and larger pieces can be inserted via the trocar . infections have occurred . to date , no postoperative bowel obstructions or readmissions for pain control or ileus have been necessary . one morbidly obese patient required urgent exploration and drainage of a large subcutaneous and intramuscular flank hematoma resulting from the use of a bladed 5-mm trocar . in the last 6 years , we have gained a great deal of practical experience with livh repair by caring for our own patients and through teaching other surgeons . we have learned that the most important details relate to ( 1 ) proper port geometry , ( 2 ) safe adhesiolysis , and ( 3 ) inserting an appropriately sized mesh to maintain adequate overlap beyond the margins of the hernia . the initial repair involved multiple widely spaced hernias covered with a single large 25 x 36-cm prosthesis . on reexplo - ration , it was obvious that the original repair was still intact and well incorporated . the recurrence was small but had occurred at the edge of the mesh closest to the working ports . inadequate prosthetic overlap on the near side was the likely cause of this preventable recurrence . incisional hernias remain a problem in many surgical specialties despite improvements in materials and adoption of methods like the mass closure technique . aside from obesity and wound infection it has been estimated that close to 10% of incisions will develop hernias . a review of 655 patients whose incisions were closed with a running monofilament utilizing a mass suturing technique revealed an incisional herniation rate of 14.2% by 2 years , with 8.4% presenting in the first year . in general , 60% to 90% of incisional hernias are observed in the first 2 to 3 years after major laparotomies . unfortunately , the recurrence rates associated with traditional open repairs are unacceptably high . a modern trial of 200 patients undergoing suture or mesh repair of a primary hernia or a first recurrence reported a 3-year recurrence rate for primary hernia repair of 43% in the suture group and 24% in the mesh group . these recurrence rates climbed to 58% when suture alone was used to close recurrent hernias . they noted that the size of the hernia did not affect the rate of recurrence and that mesh repair ( retrofascial preperitoneal polypropylene ) was superior to suture repair alone . the traditional vertical mayo or vest - over - pants repair has been shown to have a 48% recurrence rate in the first 3 years when used to repair midline incisional hernias . historically , primary tissue repairs have been associated with a 30% to 50% recurrence rate . although the adoption of tension - free repair techniques with mesh prostheses has dropped the recurrence rates to the 10% to 20% range , it is still unacceptably high . one series of 250 hernias repaired over 14 years yielded a wound complication rate of 34% . the complications being reported were infection , seroma formation , hematoma , and fistula formation . those patients in whom mesh was used during their repair suffered a wound complication rate of 49% and an infection rate of 18% . in 1987 , stoppa reported an infection rate of 18.5% in his series of 230 retrorectus mesh repairs . more recently the use of prosthetic materials in open ventral hernia repairs reduces the risk of recurrent hernias but carries the risk of increasing wound infections . it is not unreasonable to presume that the large amount of dissection and tissue undermining / devascularization required to accomplish these repairs has contributed to the infection rate . the pain associated with the dissection and mesh fixation certainly plays a role in the need to admit many of these patients to the hospital postoperatively . the improved results have been maintained despite variations in technique and prosthetics from study to study ( table 1 ) . the common denominator in all successful livh repairs is the intraperitoneal placement of the prosthesis . in general , the only things that have changed from one study to the next are the types of prostheses being inserted , the degree of hernia overlap , and the manner of fixation . we have previously demonstrated that more than 95% of these patients can be discharged from the recovery room regardless of the size of the defect . others have demonstrated a significant decrease in facility costs when comparing open and laparoscopic repairs as 90% of their laparoscopic hernia repairs did not require inpatient care . shorter hospital stays and less severe postoperative complications in laparoscopic repairs contributed to statistically significant lower costs relative to open repairs in the same study . a group of surgeons cut their hernia recurrence rate by nearly 50% when they switched from open placement of eptfe to laparoscopic placement . they declared the decrease in postoperative pain , complications , and lowered recurrence rate to be a great benefit to our practice . it is difficult to accurately catalogue the various laparoscopic techniques that have been tried in an effort to reduce recurrence rates and complications associated with traditional anterior repairs . they are often simply a reflection of the technology available at that particular time and the skill of the surgeon involved . some aspects of the repairs such as patient preparation , trocar placement , and lysis of adhesions change very little from report to report . . early attempts at laparoscopic ventral hernia repair were often a reflection of what was being taught for inguinal hernia repair . preperitoneal dissections were made and polypropylene mesh was placed over the defect and stapled to the abdominal wall . some have referred to it as being virtually prohibitive . as a result , methods for complete intraabdominal mesh placement have been evaluated . although surgeons were familiar with polypropylene mesh from open hernia repairs , many were worried about the complications that could develop from a complete intraabdominal placement of this material . in some techniques , the omentum is used to act as a barrier between the polypropylene and the bowel . in fact , many authors began to use eptfe in their repairs despite its much higher cost . they felt that the possible reduction in both intraabdominal adhesions and the risk of bowel fistualization justified the cost of the eptfe . despite these concerns , some surgeons have produced large series of patients repaired with polypropylene that have excellent results and no evidence of complications related to bowel adherence to the mesh . despite advantages in terms of intraabdominal adhesion formation , eptfe presented its own unique set of challenges for surgeons . very experienced laparoscopic surgeons have described manipulation of this mesh as a cumbersome procedure and difficult to work with laparoscopically because of its lack of memory and the fact that it is opaque . surgeons also began to realize that hernia staplers were inadequate to transfix this material to the abdominal wall and began looking for alternatives . in 1996 , none of the currently available articulating endoscopic staplers consistently and adequately secured a 1-mm ptfe patch to a depth beyond the peritoneum . he and others began to increase the length of their overlap to 3 cm or greater and to utilize full thickness transabdominal stay sutures to secure the mesh . these measures also serve to compensate for the relative lack of fibrous ingrowth into the ptfe relative to polypropylene . ptfe repairs rely heavily on the integrity of the suture attachment of the prosthesis to the fascia . failure to place these full - thickness sutures in ptfe repairs results in higher recurrence rates . when eptfe is properly sized and transfixed , excellent results can be achieved with laparoscopic ventral hernia repairs ( table 1 ) . unfortunately , some of the mesh - specific needs of eptfe can make these repairs difficult for surgeons who do not have a great deal of experience with the technique . in some cases , the proper placement and orientation of the sutures can add up to 50% to the time needed for repair . these sutures represent a potential portal for contamination of the mesh with skin flora , risking infection and possible mesh removal . reports have also been made of long - term pain associated with the sutures , and in some cases second procedures have been necessary to remove the offending suture . we have found composix mesh and now composix e / x mesh to be excellent prostheses for the laparoscopic repair of ventral and incisional hernias . their eptfe surface protects the visceral organs from significant adhesions while the polypropylene surface provides a dependable template for rapid fibroblastic ingrowth . additionally , the polypropylene layer gives the mesh enough memory to make it easy to manipulate both small and large pieces in a laparoscopic field . we feel the technique we have described for livh repair is one that can be easily replicated by most general surgeons without the need for advanced training in laparoscopic surgery . surgical technique for repair of hernias is as old as the profession itself . over time , the accepted gold standards for repair of specific subsets of hernias have been challenged and often replaced . standard techniques is often the result of technical innovation and the availability of novel materials . until the recurrence rate reaches zero and pain is eliminated from the process of hernia repair , we will continue to strive to do better . the contemporary results of open incisional and ventral hernia repair are unsatisfactory because of high recurrence rates and morbidity levels . the minimally invasive approach to repairing even complex ventral and incisional hernias is gaining rapid acceptance across the country due to its lower recurrence rate , reduced complication rates , and patient satisfaction . the location for laparoscopic repair of these hernias is quickly moving from the large academic centers to community hospitals as surgeons educate themselves on newer , less awkward techniques for mesh insertion and fixation . a gold standard for laparoscopic ventral and incisional hernia is difficult to develop when the products being used change so rapidly . to our advantage , newer - generation products ( ie , nonbladed optical trocars , ultrasonic shears , smaller tackers , and improved mesh composites ) are making these repairs safer for patients and easier to learn for surgeons who do not have the benefit of advanced laparoscopic training consequently , surgeons must keep an open mind about all of the products and techniques being offered to facilitate the repair now and in the future .

background and objectives : the contemporary results of open incisional and ventral hernia repair are unsatisfactory because of high recurrence rates and morbidity levels . laparoscopic repair of ventral and incisional hernias ( livh ) can be accomplished in a simple , reproducible manner while dramatically lowering recurrence rates and morbidity.methods:one hundred consecutive patents underwent laparoscopic repair of their ventral and incisional hernias over a 27-month period . composix mesh and composix e / x mesh ( davol inc . , cranston , ri ) were utilized for the repairs . transfixion sutures were not used.results:all repairs were completed laparoscopically . no conversions to open techniques were necessary . no postoperative infections have been observed . one recurrent hernia was identified and subsequently repaired with the same technique.conclusions:livh can be accomplished with a dramatic reduction in recurrence rates and morbidity . the technique for this repair is still in a state of evolution . the construction and handling characteristics of this particular type of mesh have allowed us to eliminate transfixion sutures and to simplify the repair technique while maintaining a very low recurrence rate .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS

PMC3692190

schwannoma is a benign nerve sheath tumor originating from schwann cells , and is known as neurilemmoma and neurinoma . although the head and neck region is a rather common site for these benign lesions , intraoral schwannomas , especially the intraosseous types , are very rare , and can account for less than 1% of the benign intraosseous neoplasms . the clinical and radiographic findings of intraosseous schwannoma may be similar to many other lesions such as odontogenic cysts and other benign tumors . therefore , various differential diagnoses have been proposed , which may lead to some sort of confusion in its diagnosis . according to the observation in the literature , these lesions are more common in the mandible and among females , which are mostly observed in the 2 and 3 decades of life . here , along with a review of the related literature , we try to present a case of intraosseous schwannomas that has been recognized in a 9-year - old boy . the patient was a 9-year - old boy who was referred to the department of oral and maxillofacial pathology of the isfahan university of medical science 5 months ago , while complaining about a swelling in the inferior border of the right mandible . deviation of anterior teeth and looseness of teeth ( d , e ) were seen . in the panoramic radiograph , radiolucency with sclerotic border from mesial of tooth 36 to distal of 3c and transposition of first and second impacted premolar was observable [ figure 1 ] . radiolucency with sclerotic border from mesial of tooth 36 to distal of 3c in the panoramic radiogeraph axial computed tomography scan revealed a unilacular lesion in the left mandibular body , 2.5 3 cm in size , with a well - defined border , marked expansion and thinning of the buccal and lingual cortical plates [ figure 2 ] . unilacular lesion in the left mandibular body , 2.5 3 cm in size , with well - defined border in axial ct scan first , on the basis of clinical and radiographic features , we can suggest the following lesions : keratocystic odontogenic tumor , central giant cell granuloma , unicystic ameloblastoma and ameloblastic fibroma . excisional biopsy was performed and irregular masses of solid tissues were observable during biopsy . in the histopathology features , ovoid to spindle shape and epithelioid cells were identified , some of which indicated slight pleomorphism and hyperchromatism besides , some regions showed intracellular edema and degenerative changes . in some parts , numerous blood vessels and perivascular hyalinization antoni a : palisading arrangement of spindle cell , verocay body , antoni b : spindle cells haphazardly distributed in a delicate fibrillar matrix . h and e : original maginifiction ( 40 ) based on histopathologic features , several differential diagnoses were first taken into account : benign fibroblastic tumor , benign neural tumors and neoplastic vascular lesions . in order to reach final diagnosis , immunohistochemical staining was carried out . the findings revealed that the intensity of s100 staining was 4 + and those of ki67 was weak and 10% at most [ figure 4 ] . according to these studies and immunohistochemistry , and regarding clinical and radiographic features and hematoxylin and eosin , in which there were some regions similar to antoni a with palisading cells and similarities of some regions to antoni b , we reached the final and definite diagnosis of the lesion . original maginifiction ( 400 ) after 4 months , the patient was evaluated by taking panoramic radiography , and it seems that reparative process is running within the lesion [ figure 5 ] . although schwannoma is a benign and slow growing tumor , and it is usually an asymptomatic one , almost 50% of the cases have been reported with pain . the most common site of this tumor in the head and neck region is the tongue . moreover , the intraosseous type is extremely rare , and accounts for less than 1% of all primary bone tumors . average age of the people afflicted is 34 years , and the male to female ratio is 1:1.5 . lesions are more common in the mandible than in the maxilla , and the swelling usually involves the body of the mandible . so far , fewer than 50 cases have been recorded in the related literature , of which 44 cases have been reported by chi et al . in this report , the clinical features of these cases have been described , and only one case was younger than 10 years old . our case is a 9-year - old boy , which appears to be extremely rare . schwannoma can be originating from all cranial nerves , with the exception of the olfactory and optic nerves . the tumor presents an expansive growth pattern , which may cause dislocation of teeth and may also involve the nerves . these above - mentioned features were also observed in the case under investigation in this study . but , the problem was that due to the scarcity of such cases , it was not possible for us to predict the schwannoma in differential diagnosis at first . in microscopic features , schwannoma is an encapsulated tumor that shows two microscopic patterns : antoni a and antoni b. streaming fascicles of spindle - shaped schwann cells characterize antoni a tissue . these cells often form a palisaded arrangement around the central acellular , eosinophilic areas , identified as verocay bodies . antoni b pattern is less cellular and the spindle cells are randomly arranged within a loose , myxomatous stroma . schwannoma indicates the positivity of s100 , cd34 and ema only in capsules of the tumor . in our case the lesion showed tumoral appearance , which was completely different from the characteristic structures of odontogenic cysts or tumors . also , specific types of cell , such as giant cells , were not obvious . the lesion showed spindle cell pattern in most of the places , which was similar to fibroblastic or neural cells . immunohistochemistry revealed the tumor to be positive for the s100 protein , confirming nerve origin . on the basis of schwannoma , two characteristic patterns : observing typical limited area similar to antoni a despite verocay body was not clearly seen , and other regions akin to antoni b pattern , the diagnosis of schwannoma was made . the present of these two important histopathologic patterns , lack of mast cells , which is one of the most helpful features in neurofibroma and also diffuse , strongly positively tumoral cells s100 expression in ihc slides , the diagnosis of nurofibroma was rejected . it is probable that some malignant changes in schwannoma may happen , even though some malignant cases have been reported . these changes consist of cyst formation , necrosis , hyalinization , hemorrhage , hemosiderin deposits , inflammation , fibrosis and nuclear atypia . nonetheless , these tumors are benign and atypical mitosis are never seen , and the pathologist must be cautious not to mistake these changes for evidence of a malignancy . also , it is possible to remove the involved nerve in order to decrease the risk of recurrence . because this lesion can be misdiagnosed clinically , radiographically and microscopically as periapical lesion , ameloblastoma , myxoma and central giant cell granuloma , careful attention should be directed toward reaching the diagnosis . only then can the appropriate treatment can be obtained .

intraosseous schwannomas is a very rare neoplasm , and less than 50 cases have been reported in the medical literature . in this article , the clinical , radiographic and histopathologic appearances of a rare case of intraosseous schwannomas are presented . the importance of this case is that other benign central lesions such as odontogenic tumors and cysts might be included in differential diagnosis . this case was recognized in a 9-year - old child , which is a very rare occurrence . the diagnosis was confirmed by immunohistochemical staining with s100 protein .

INTRODUCTION CASE REPORT DISCUSSION CONCLUSION

PMC4303325

the tetraazamacrocyclic architecture of the tropocoronand ligand provides a flexible scaffold that can accommodate varied coordination environments and stabilize unusual or otherwise unachievable geometries at transition - metal centers ( chart 1 ) . in the absence of entatic tuning , cobalt(ii ) metal centers display a proclivity toward high - spin octahedral or tetrahedral configurations , and cobalt(iii ) centers prefer low - spin octahedral geometries . square - planar coordination environments are also readily observed for four - coordinate cobalt(iii ) complexes , but small molecules with cobalt(iii ) centers in tetrahedral environments are significantly scarce . the cobalt(iii ) center in [ cow12o40 ] exists in a tetrahedral environment , as does cobalt(iii ) in [ co(nor)4 ] , where nor is the norbornyl anion . a small number of mononuclear cobalt(iii ) imides have been prepared and structurally characterized , and in these species , the multiple - bond character of the metal the reason for the rarity of cobalt(iii ) in tetrahedral environments becomes clear upon comparison of orbital splitting diagrams for a d metal center in square - planar versus tetrahedral geometries . the orbital filling diagram for a square - planar d center is shown in figure 1 for an s = 1 electronic configuration . the allocation of electrons corresponds to minimization of electron repulsion interactions and is the observed electronic distribution in [ co(tc-4,4 ) ] complexes . in contrast , the orbital distribution in a d tetrahedral orbital splitting diagram results in the partial occupancy of two destabilized t2 molecular orbitals , with no contribution to minimization of electron repulsion interactions . that is , the transition from square - planar to tetrahedral geometry results in partial filling of orbitals that are destabilized in the tetrahedral configuration relative to the square - planar one , without minimizing electron repulsion . the square - planar configuration also minimizes the electron ligand repulsion from the dx y orbital . orbital correlation diagram for the transition from square - planar to tetrahedral geometry , shown for the electron occupancy corresponding to a d metal center . modified from albright , burdett , and whangbo . having the ability to control the environment at the metal center simply by changing the tropocoronand linker chain length places us in a unique situation to examine unusual coordination environments . the previously established series of four - coordinate cobalt(iii ) tropocoronand complexes comprises [ co(tc-3,3)](x ) ( x = bph4 , bar4 ) and [ co(tc-4,4)](bph4 ) . these species exist in square - planar or distorted square - planar geometries , with twist angles , defined as the angle between planes formed by the metal and the two sets of aminotroponeiminate nitrogen atoms , of 8 and 41 for the four - coordinate complexes . in our pursuit to examine the size dependence of cobalt(ii ) and cobalt(iii ) tropocoronands on their ability to tune reactivity with nitric oxide , we prepared and structurally characterized two pseudotetrahedral cobalt(iii ) complexes , [ co(tc-5,5)](bf4 ) and [ co(tc-6,6)](bph4 ) . handling of air- and moisture - sensitive materials was conducted in an mbraun glovebox under a nitrogen atmosphere . methylene chloride and tetrahydrofuran ( thf ) solvents were purified by passage through activated alumina and stored over 4 molecular sieves under a nitrogen atmosphere prior to use . deuterated nmr solvents were obtained from cambridge isotope laboratories , stored under an inert nitrogen atmosphere , and used without further purification . the syntheses of [ co(tc-5,5 ) ] , [ co(tc-6,6 ] , [ zn(tc-5,5 ) ] , and [ zn(tc-6,6 ) ] are described elsewhere . fc(bph4 ) was prepared according to previously published procedures . to a solution of [ co(tc-5,5 ) ] ( 150 mg , 0.35 mmol ) in methylene chloride was added fc(bf4 ) ( 94.4 mg , 0.346 mmol ) . the solution was evaporated to dryness and the resultant solid was washed with diethyl ether ( et2o ) to remove ferrocene . recrystallization from dichloromethane ( dcm)/et2o at 30 c yielded x - ray - quality crystals ( 174 mg , 97% yield ) . uv vis nir [ cdcl3 ; , nm ( , m cm ) ] : 349 ( 13650 ) , 412 ( 15670 ) , 757 ( 11420 ) , 1166 ( 9183 ) . calcd for c24h30bcof4n40.32ch2cl2 : c , 53.35 ; h , 5.64 ; n , 10.23 . found : c , 53.33 ; h , 5.89 ; n , 10.21 . evidence for ch2cl2 appeared in the nmr spectrum taken in cd2cl2 . to a solution of [ co(tc-6,6 ) ] ( 200 mg , 0.4 mmol ) in methylene chloride was added fc(bph4 ) ( 218 mg , 0.434 mmol ) in the dark . the solution was evaporated to dryness , and the resultant solid was washed with et2o to remove ferrocene . recrystallization from dcm / et2o at 30 c yielded x - ray - quality crystals ( 330 mg , 97% yield ) . note : we found methylene chloride solutions of fc(bph4 ) to be unstable and attribute variations in product yield to this property . nir [ cdcl3 ; , nm ( , m cm ) ] : 271 ( 60900 ) , 349 ( 21470 ) , 424 ( 34560 ) , 702 ( 2362 ) , 981 ( 561 ) , 1240 ( 475 ) . calcd for c50h54bcon4 : c , 76.92 ; h , 6.97 ; n , 7.18 . found : c , 76.68 ; h , 6.71 ; n , 7.33 . to a solution of h2tc-5,5 ( 300 mg , 0.8 mmol ) in thf was added nahmds ( 292 mg , 1.59 mmol ) , and the reaction was allowed to stir for 10 min . gacl3 ( 448 mg , 1.59 mmol ) was added to the solution as a solid , and the reaction was left to stir overnight . the solution was evaporated to dryness , suspended in ch2cl2 , and filtered through celite . recrystallization from ch2cl2/et2o at 30 c yielded x - ray - quality crystals ( 370 mg , 70% yield ) . h nmr ( cd2cl2 ) : 1.68 ( m , 8h , ch2 ) , 1.91 ( m , 4h , ch2 ) , 3.74 , ( m , 4h , ch2 ) , 3.85 ( m , 4h , ch2 ) , 7.10 ( t , j = 10 hz , 2h , arh ) , 7.26 ( d , j = 8 hz , 2h , arh ) , 7.64 ( t , j = 12 hz , 2h , arh ) . esi - ms ( [ m gacl4 ] ) : m / z 433.1 ( calcd m / z 433.12 ) . uv vis [ cdcl3 ; , nm ( , m cm ) ] : 274 ( sh , 63390 ) , 280 ( 70810 ) , 365 ( 40920 ) , 416 ( sh , 19480 ) , 433 ( 27090 ) . calcd for c24h30n4ga2cl4 : c , 43.96 ; h , 4.61 ; n , 8.54 . found : c , 43.88 ; h , 4.44 ; n , 8.42 . to a solution of h2tc-6,6 ( 54 mg , 0.13 mmol ) in thf was added nahmds ( 45 mg , 0.27 mmol ) , and the reaction was allowed to stir for 15 min . gacl3 ( 70 mg , 0.27 mmol ) was added to the solution as a solid , and the reaction was left to stir overnight . the solution was evaporated to dryness , suspended in ch2cl2 , and filtered through celite . recrystallization from ch2cl2/et2o at 30 c yielded x - ray - quality crystals ( 52 mg , 57% yield ) . h nmr ( cd2cl2 ) : 1.131.26 ( m , 8h , ch2 ) , 1.44 ( m , 4h , ch2 ) , 2.192.26 ( m , 4h , ch2 ) , 3.643.71 ( m , 4h , ch2 ) , 3.974.02 ( m , 4h , ch2 ) , 7.12 ( t , j = 8 hz , 2h , arh ) , 7.35 ( d , j = 8 hz , 2h , arh ) , 7.60 ( t , j = 6 hz , 2h , arh ) . esi - ms ( [ m gacl4 ] ) : m / z 471.1 ( calcd m / z 471.20 ) . vis [ cdcl3 ; , nm ( , m cm ) ] : 272 ( sh , 73750 ) , 280 ( 83830 ) , 363 ( 45720 ) , 410 ( sh , 22980 ) , 422 ( sh , 28930 ) , 429 ( 37980 ) . ir ( kbr ; cm ) : c = n 1514 , calcd for c26h34n4ga2cl4 : c , 45.67 ; h , 5.01 ; n , 8.19 . evan s method measurements were made in cd2cl2 on a 500 mhz varian inova spectrometer , and the temperature was measured by the residual peak separation of the h nmr of neat cd3od . optical spectra were recorded on a varian cary 5000 uv vis nir spectrophotometer in 6sq starna cells . fourier transform infrared spectra were recorded on a thermo nicolet avatar 360 spectrometer running the omnic software package . electrospray ionization mass spectrometry ( esi - ms ) analyses were performed on the agilent 1100 series lc / msd trap spectrometer . cyclic voltammograms were recorded under nitrogen using the versastat3 potentiostat ( princeton applied research ) and v3 studio software . a glassy carbon working electrode , samples were prepared as 35 mm solutions in methylene chloride with 0.1 m ( n - bu4n)(pf6 ) as the supporting electrolyte . reported spectra the reversible fc / fc couple appeared at 0.57 v vs ag / ag . crystals were mounted in paratone n oil and frozen at 100 k under a cold nitrogen stream controlled by a cryopad low - temperature apparatus . data were collected on a bruker apex ccd x - ray diffractometer with graphite - monochromated mo k radiation ( = 0.71073 ) controlled by the apex2 software package . the structure was solved by direct methods using shelxs-97 and refined by full - matrix least squares on f using the shelxl-97 program incorporated into the shelxtl software package . hydrogen atoms were assigned idealized positions and given thermal parameters 1.2 times the thermal parameters of the atoms to which they are attached . the structure of [ ga(tc-6,6)](gacl4 ) contained voids filled with heavily disordered solvent molecules . the program squeeze(28 ) was used to remove the contributions of the disordered solvent to the structure factors . the electron density attributed to disordered solvent molecules created a channel along the 65 screw axis and corresponded to seven molecules of methylene chloride or et2o . the crystal of [ ga(tc-6,6)](gacl4 ) was an inversion twin , and the percentage of the main twin component was refined to 58.2% . [ co(tc-5,5 ) ] was successfully oxidized to [ co(tc-5,5)](bf4 ) by reaction with ferrocenium tetrafluoroborate in dcm ( figure 2 , left , and tables s1 and s2 in the supporting information , si ) . structural characterization of [ co(tc-5,5)](bf4 ) revealed the twist angle at cobalt to be 65. the average bond distance between the metal center and coordinating nitrogen atoms , co1nave , is 1.85 , comparable to the 1.86 and 1.87 values in [ co(tc-3,3)](bph4 ) and [ co(tc-4,4)](bar4 ) , respectively . thermal ellipsoid plots for [ co(tc-5,5)](bf4 ) ( left ) and [ co(tc-6,6)](bph4 ) ( right ) , shown at 50% probability . we also oxidized [ co(tc-6,6 ) ] to [ co(tc-6,6)](bph4 ) with ferrocenium tetraphenylborate and structurally characterized the resulting cobalt(iii ) product ( figure 2 , right , and figure s2 and tables s1 and s3 in the si ) . the twist angle in [ co(tc-6,6 ) ] is 74 , the largest value observed for cobalt(iii ) tropocoronands to date . the co1nave bond distance is 1.82 , somewhat shorter than that in previously reported cobalt(iii ) tropocoronand coordination compounds . a comparison of bond lengths and twist angles for four - coordinate cobalt(ii ) and -(iii ) tropocoronand complexes is provided in table 1 . tetrahedral geometry is rare for cobalt(iii ) and , to our knowledge , has been observed in mononuclear small molecules only for [ co(nor)4 ] and a handful of cobalt(iii ) imido complexes to date . the paucity of cobalt(iii ) in tetrahedral environments and the unusual geometries of the metal centers in [ co(tc-5,5 ) ] and [ co(tc-6,6 ) ] raised the possibility that oxidation of the parent cobalt(ii ) compounds occurred at the ligand rather than the metal center . we therefore prepared and characterized [ ga(tc-5,5)](gacl4 ) and [ ga(tc-6,6)](gacl4 ) and compared their structural and electrochemical properties with those of the analogous cobalt complexes ( figure 3 and tables s1 , s4 , and s5 in the si ) . thermal ellipsoid plots for [ ga(tc-5,5)](gacl4 ) and [ ga(tc-6,6)](gacl4 ) , depicted at 50% probability . the [ ga(tc-5,5)](gacl4 ) complex crystallizes in p21/c and exhibits crystallographic disorder over the entirety of the tropocoronand ligand ( figure s1 in the si ) . the n4-coordinated gallium(iii ) center has a twist angle of 81 , displaying distorted tetrahedral geometry . the average gallium [ ga(tc-6,6)](gacl4 ) crystallizes in p65 and exhibits nearly perfect tetrahedral coordination at the gallium(iii ) center . the twist angle in [ ga(tc-6,6 ) ] comparison of analogous bond distances between the [ co(tc - n , n ) ] and [ ga(tc - n , n ) ] complexes provided insight into the electron distribution in the former species ( figures 4 and 5 ) . nitrogen bonds in both cobalt complexes are shorter than those in the analogous gallium(iii ) tropocoronands . n bond is probably a consequence of the greater covalent character of the cobalt tropocoronand compared to the gallium tropocoronand complex . comparison of structural parameters for [ co(tc-5,5)](bf4 ) ( left ) and [ ga(tc-5,5)](gacl4 ) ( right ) . distances ( ) shown are representative of bond distances within the tropocoronand ligand . comparison of structural parameters for [ co(tc-6,6)](bph4 ) ( left ) and [ ga(tc-6,6)](gacl4 ) ( right ) . distances ( ) shown are representative of bond distances within the tropocoronand ligand . in a review of the experimental and theoretical properties of transition - metal complexes bound to redox noninnocent ligands , ray et al . note difficulties in using x - ray structural parameters to draw conclusions regarding ligand- versus metal - based oxidation in delocalized systems , where the electron of a ligand radical is shared between two different ligands . the tropocoronand complexes provide such a delocalized system , for if a ligand radical were to form , the electron would be able to travel between the two aminotroponeiminate rings via the metal center . we are also aware of reports describing noninnocent ligands that undergo negligible structural rearrangement upon changes in redox state . we therefore turned to electrochemical methods for further evidence that oxidation of [ co(tc-5,5 ) ] and [ co(tc-6,6 ) ] results in cobalt(iii ) species . previously published cyclic voltammetry studies of [ co(tc-5,5 ) ] and [ co(tc-6,6 ) ] were repeated and compared to the results of analogous studies of [ ga(tc-5,5)](gacl4 ) and [ ga(tc-6,6)](gacl4 ) ( figure 6 ) . we observed reversible couples at 0.344 v vs fc / fc for [ co(tc-5,5 ) ] and 0.367 v vs fc / fc for [ co(tc-6,6 ) ] . we assign these processes to metal - based redox reactions by comparison with electrochemical studies of [ zn(tc-5,5 ) ] and [ zn(tc-6,6 ) ] . the absence of similar reversible processes in the voltammograms of the zinc complexes is consistent with these redox events being metal - based . additionally , we assign the irreversible features at 0.467 v in the [ co(tc-5,5 ) ] voltammogram and at 0.646 v in the [ co(tc-6,6 ) ] voltammogram to ligand - based oxidations . the cobalt(ii)/cobalt(iii ) couples observed here appeared at 100 mv more negative than those previously published . the reason for this discrepancy is unknown , and the current values are considered to be correct . cyclic voltammetry of [ ga(tc-5,5)](gacl4 ) and [ ga(tc-6,6)](gacl4 ) revealed ligand - based oxidations at 0.846 and 0.920 v vs fc / fc , respectively . ligand oxidation occurred at more positive potentials in the gallium(iii ) complexes than in the analogous zinc(ii ) and cobalt(iii ) compounds . the shift to more positive potentials in the gallium(iii ) tropocoronands relative to the zinc(ii ) analogues can be attributed to the higher oxidation state of the gallium center . ligand oxidation in the gallium complexes may occur at more positive potentials than in the corresponding cobalt species because the cobalt center may be better able to stabilize the additional charge through covalent metal ligand interactions than gallium . cyclic voltammograms of ( a ) [ co(tc-5,5 ) ] , ( b ) [ co(tc-6,6 ) ] , ( c ) [ ga(tc-5,5)](gacl4 ) , and ( d ) [ ga(tc-6,6)](gacl4 ) , referenced to fc / fc . we attempted to characterize [ co(tc-5,5)](bf4 ) by x - band electron paramagnetic resonance ( epr ) spectroscopy but were unable to observe any signal at 77 k. helium temperature epr spectroscopic studies of [ co(tc-6,6)](bph4 ) were equally unrevealing . the h nmr spectrum of [ co(tc-5,5)](bf4 ) shows broad , low - intensity peaks in the diamagnetic region of the spectrum , which may correspond to a tropocoronand - containing species ( figure s3 in the si ) . peaks corresponding to protons of the tropocoronand ligand are absent in the h nmr spectrum of [ co(tc-6,6)](bph4 ) , but peaks for the tetraphenylborate counteranion are readily observed ( figure s4 in the si ) . previous studies revealed that , as the total length of the linker chains ( n + m ) of a ( tc - m , n ) complex increases , both the geometry of the metal center and spin state change . the spin state of the distorted square - planar [ ni(tc-4,5 ) ] is s = 0 , and that of the distorted tetrahedral [ ni(tc-5,5 ) ] is s = 1 . in the cobalt(ii ) system , a spin - state change is observed between [ co(tc-4,4 ) ] ( s = /2 ) and [ co(tc-4,5 ) ] ( s = /2 ) . we previously reported that [ co(tc-3,3)](bph4 ) ( twist angle = 8 ) and [ co(tc-4,4)](bph4 ) ( twist angle = 41 ) have spin state s = 1 at room temperature and magnetic moments of eff = 3.1 and 3.6 b , respectively . to determine whether a spin - state change occurs in the [ co(tc - m , n ) ] series , the magnetic susceptibility of [ co(tc-6,6)](bph4 ) was measured by evan s method at room temperature . the magnetic moment was determined to be 5.38 b , as expected for s = 2 , confirming that a spin - state change does occur . calculations were performed with the use of orca(40 ) to supplement our understanding of the electronic structure of the [ co(tc - n , n ) ] series ( n = 36 ) . in all cases , the optimized geometries [ bp / svp(tzvp on co ) ] of the s = 2 state had twist angles 1321 larger than that those of the s = 1 state ( table s6 in the si ) . this result supports the connection between the spin - state change and the square - planar to tetrahedral change in geometry . subsequent energy computations at the b3lyp / def2-tzvp level predicted that the s = 1 state is more stable than the s = 2 state in all four species , with triplet quintet gaps of 24.9 , 18.9 , 8.0 , and 7.2 kcal / mol for n = 36 , respectively ( figure s6 in the si ) . our experimental results show that a spin - state change to s = 2 occurs for n = 5 or 6 . the computations suggest that the ground - state electronic structure for n = 5 and 6 is s = 1 , but there is a low energy barrier to accessing the s = 2 state ( 78 kcal / mol ) . even though this level of theory incorrectly predicts the ground state spin for n = 5 and 6 , the trend in the tripletquintet energy gap across the series n = 36 is correct . quintet energy gap correlates almost linearly with the experimental twist angle ( r = 0.936 ; figure s6 in the si ) . electronic transitions predicted for the series support the spin - state change but are less conclusive ( figures s7s10 in the si ) . molecular orbitals involved in the electronic transitions for all complexes contained significant mixing between the cobalt and tropocoronand orbitals . thus , assignments for the transitions in the electronic spectra could not be made without further work . comparison of structural and electrochemical properties of the cobalt and gallium species [ co(tc-5,5 ) ] , [ co(tc-6,6 ) ] , [ ga(tc-5,5 ) ] , and [ ga(tc-6,6 ) ] confirmed the cobalt(iii ) character of the metal centers in [ co(tc-5,5)](bf4 ) and [ co(tc-6,6)](bph4 ) . the synthesis of [ co(tc-5,5)](bf4 ) and [ co(tc-6,6)](bph4 ) augments the number of cobalt(iii ) species having the rare pseudotetrahedral geometry . together with previous results from our laboratory , the present study reveals that a spin - state change from s = 1 to s = 2 occurs as the ligands tune the geometry of the complexes from pseudo - square - planar for [ co(tc - n , n)](bph4 ) ( n = 3 , 4 ) to pseudotetrahedral for [ co(tc-6,6)](bph4 ) .

the preparation and characterization of two mononuclear cobalt(iii ) tropocoronand complexes , [ co(tc-5,5)](bf4 ) and [ co(tc-6,6)](bph4 ) , are reported . the cobalt(iii ) centers exist in rare pseudotetrahedral conformations , with twist angles of 65 and 74 for the [ co(tc-5,5]+ and [ co(tc-6,6)]+ species , respectively . structural and electrochemical characteristics are compared with those of newly synthesized [ ga(tc-5,5)](gacl4 ) and [ ga(tc-6,6)](gacl4 ) analogues . the spin state of the pseudotetrahedral [ co(tc-6,6)](bph4 ) compound was determined to be s = 2 , a change in spin state from the value of s = 1 that occurs in the square - planar and distorted square - planar complexes , [ co(tc-3,3)](x ) ( x = bph4 , bar4 ) and [ co(tc-4,4)](bph4 ) , respectively .

Introduction Experimental Methods Results and Discussion Summary and Conclusions

PMC3711909

multi - frequency bia is described as a tool able to assess total , extracellular and intracellular fluid in humans [ 14 ] , but its ability to diagnose water - loss dehydration has not been previously assessed . water - loss dehydration , signified by raised serum osmolality and due to insufficient fluid intake and sometimes increased output is common after stroke and associated with increased mortality and morbidity [ 58 ] . dysphagia , depression and concern over continence all increase the risk of dehydration , and stroke is more common in those who are already dehydrated . hydration status can alter rapidly in people not drinking adequately , so monitoring hydration status in this particularly vulnerable group is important especially in non - hospital settings such as residential and nursing homes . serum osmolality , the best reference standard for water - loss ( or hypertonic ) dehydration is the osmolar concentration or osmotic pressure of serum , reflecting the concentration of dissolved particles per kilogram of serum . serum osmolality reflects the osmolality of intracellular fluid , and thus cell volume , as most cell walls are permeable to water allowing osmolality to equalise between the intra- and extra - cellular fluid . as osmolality is carefully controlled by the body , any change suggests important alterations in cellular hydration . serum osmolality has advantages as a reference standard for dehydration as it can be used alone , and without prior measurement . weight change has been used as a marker for dehydration but requires at least 2 measurements ( so can not be used on an unknown patient ) , and can be masked by changes in other body components ( fat and muscle ) as well as mimicked or masked by constipation and oedema . thus serum osmolality has the best easily measurable characteristics of a reference standard for water - loss dehydration and is the diagnostic standard against which the accuracy of other measures should be judged . the dehydration council s cut - off points for serum osmolality are specific to water - loss dehydration in older people . serum osmolality of 295300 mosm / kg equates to impending dehydration , and > 300 mosm / kg to current dehydration , definitions used in this study . in clinical practice serum osmolality is often not directly measured , but calculated from the combined concentrations of serum sodium , potassium , glucose and urea , referred to as serum osmolarity ( [ 2na]+[2k]+urea+glucose , all in mmol there is a small difference between measured serum osmolality and calculated osmolarity , known as the osmolar gap ( as some components of osmolality are not included in the formula to calculate osmolarity ) . methods to assess hydration status which do not require blood samples would be helpful for use in those who have had a stroke once they leave hospital , especially in situations where there is no quick and easy access to laboratory facilities such as rehabilitation services , care homes and the community . there is some evidence that signs used to indicate water - loss dehydration in older people such as urinary and physical assessments are unreliable and inaccurate . while single frequency bioelectrical impedance assumes full hydration and so is unable to assess hydration status , multi frequency bioelectrical impedance analysis ( mf - bia ) unlike single frequency bia , can provide estimates of total body water ( tbw ) , intracellular water ( icw ) , and extracellular water ( ecw ) volumes and as percentages of body weight . this is because the 5 khz frequency can estimate ecw , while frequencies over 50khz predict tbw ( and icw = tbw - ecw ) . the maltron website states the bioscan 920 - 2s multi - frequency analyser with its unique features is a rapid , non - invasive , inexpensive method for evaluating hydration and nutrition status the advance ( sic ) circuitry and processing power of the bioscan 920 - 2s allows it to measure extracellular ( ecf ) and intracellular fluid ( icf ) volume ( see www.maltronint.com/products/bioscan920-2s.php , accessed 6/11/2012 ) . despite this claim of evaluating hydration status , the diagnostic accuracy of mf - bia in diagnosis of dehydration has not previously been published to our knowledge . this study aimed to assess the diagnostic accuracy of mf - bia , to help understand whether mf - bia can be used to monitor hydration status in place of serum osmolality after stroke . this prospective study was carried out in the norfolk and norwich university hospital in the east of england . twenty seven stroke patients admitted within 48 hours of symptom onset were recruited from 1 april to 15 oct 2011 . patients were included if older than 17 years , with newly diagnosed stroke ( first or recurrent ) . exclusions included those with severe stroke ( national institute of health stroke scale , nihss , score > 30 ) , co - existing terminal illness , expected survival < 48 hours as judged by a stroke physician , or inability or unwillingness to give informed consent . all eligible patients who provided informed consent during the study period were recruited . upon consent a venous blood sample was taken for serum osmolality , sodium , potassium , random glucose , urea and creatinine and analysed by the hospital pathology laboratory immediately . serum osmolality was analysed using freezing point depression on an advanced instruments model 2020 osmometer ( advanced instruments inc , massachusetts 02062 usa ) , and all other measures were made on an abbott c8000 analyser ( abbott laboratories , abbott park , illinois 60064 , usa ) standardised and automated . glucose was measured using a hexokinase based enzymatic method ( reagent serial number 3l82 - 20 ) , sodium and potassium using an indirect ion sensitive electrode ( reagents were ict sample diluent 2p32 , and ict reference solution 1e49 ) , creatinine was measured using an alkaline picrate reagent system . all reagents were supplied by abbot , the reagents kit serial number was 3l81 - 31 . participants removed any jewellery , and were asked if they would like to micturate before the mf - bia measurement was carried out . two consecutive mf - bia measurements ( bioscan 920 - 2 , maltron international ltd , essex ; using brand new equipment ) were taken within 20 minutes of the blood sampling with the subject supine , before serum osmolality results were available ( the assessor was blinded to hydration status ) . mf - bia measurements were undertaken using the manufacturers recommended method with two electrodes attached to the skin between the talus and the 3 and 5 digits of the foot and two more attached to the same side between the 3 and 5 knuckles of the hand and the wrist . patients were weighed using a chair scale ( seca 955 electronic scale , seca scales and measuring systems , birmingham , uk ) , and those unable to stand were weighed using a hoist ( loco - motor multi - lift hoist , select healthcare , northants , uk ) . height was estimated from ulna length ( measured using the distance between the olecranon and styloid processes of the left hand ) . weight , height , age , gender , and race were entered into the device and the mf - bia measurements made over a couple of seconds . after measurements were recorded and saved , data were downloaded onto a laptop using maltron mf - bia software ( mistat 920 ) . impedances at 5 , 50 and 100 khz and mf - bia calculations of total body water as a percentage of body weight ( tbw% ) , intracellular water as a percentage of tbw ( icw% ) and extracellular water as a percentage of tbw ( ecw% ) were noted for each recording . modified rankin scores ( mrs , a measure of disability ) were recorded by an occupational therapist . ethical approval for this study was gained from cambridgeshire i research ethics committee ; rec reference number 10/h0304/18 in april 2011 . all statistical analyses were carried out using pasw 18 for windows ( polar engineering and consulting , formerly known as spss ) . mean , standard deviation ( sd ) and range were presented for continuous and numbers ( percentages ) were presented for categorical data . percentages of patients diagnosed with impending ( serum osmolality 295300 mosm / kg ; serum osmolarity 295300 mosm / l ) and current dehydration ( serum osmolality > 300 mosm / kg ; serum osmolarity > 300 a paired sample student s t - test was carried out to compare the difference in serum osmolality and calculated osmolarity values overall and stratified by hydration status ; hydrated , impending , and current dehydration . an average was calculated for each two consecutive measurements taken by mf - bia of same variable for use in subsequent calculations . for the one participant where the two consecutive estimates of tbw% varied by > 3% the first data set was used . the internal consistency of mf - bia was assessed by carrying out a reliability analysis of the 2 separate measurements of impedance at 5 khz for each individual . this was repeated for impedance measures at 50 and 100 khz , and the mf - bia equipment calculation of tbw ( l ) . impedance outputs ( mean from the two readings ) were used to calculate tbw ( l ) and ecw ( l ) using equations developed for use in older people ( mean age 67 , similar to our participants ) by vach and visser ( as quoted in ritz ( 22 ) ) , and tbw% , ecw% and icw% were calculated as percentages of body weight1 . total body water ( bw ) was calculated using vach equations ( 19 ) and used in estimating tbw : where r100 is impedance at 100hz , g is gender ( 0 for women and 1 for men ) and height and weight are measured in meters and kilograms . extracellular water was estimated using equations by visser ( as quoted in ritz ): where r5 is impedance at 5hz . tbw% , ecw% , icw% and ecw : icw ratio from the internal calculations of the mf - bia equipment , and those calculated from equations derived specifically for older people were each plotted in 22 tables against impending and current serum osmolality and calculated serum osmolarity . these tables were used to calculate sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) , pre- and post - test probability of each for impending and current dehydration . where any of these values were not calculable due to the presence of zeros in the 22 table , 0.1 was added to each cell of the table . as published cut - off points of tbw , ecw and icw for dehydration are not readily available , three arbitrary cut - off points were selected for each measure ( tbw% , ecw% , icw% and the ratio ) . receiver operating characteristic ( roc ) curves were created for both impending and current dehydration , then additional promising cut - off points ( where cut - offs may possibly have both sensitivity and specificity > 60% ) were added to fill in the roc curves . at the ends of the roc curve , once either sensitivity or specificity was below 50% , no further outlying points were added . an acceptable cut - off point was considered to be one with both sensitivity and specificity greater than 60% and represented by the point closest to the top left corner of the roc plot . there is no definition of good enough sensitivity and specificity but we chose a minimum of 60% for both as suggesting that the measure was at least promising . for all cut - off points we also calculated positive predictive value ( ppv ) , negative predictive value ( npv ) , and positive and negative post - test probabilities . no sample size calculation was performed as there were no data available previously reporting diagnostic accuracy of mf - bia against serum osmolality . twenty seven participants were a realistic sample given the time frame we were able to use for this study . all statistical analyses were carried out using pasw 18 for windows ( polar engineering and consulting , formerly known as spss ) . mean , standard deviation ( sd ) and range were presented for continuous and numbers ( percentages ) were presented for categorical data . percentages of patients diagnosed with impending ( serum osmolality 295300 mosm / kg ; serum osmolarity 295300 mosm / l ) and current dehydration ( serum osmolality > 300 mosm / kg ; serum osmolarity > 300 was carried out to compare the difference in serum osmolality and calculated osmolarity values overall and stratified by hydration status ; hydrated , impending , and current dehydration . an average was calculated for each two consecutive measurements taken by mf - bia of same variable for use in subsequent calculations . for the one participant where the two consecutive estimates of tbw% varied by > 3% the first data set was used . the internal consistency of mf - bia was assessed by carrying out a reliability analysis of the 2 separate measurements of impedance at 5 khz for each individual . this was repeated for impedance measures at 50 and 100 khz , and the mf - bia equipment calculation of tbw ( l ) . impedance outputs ( mean from the two readings ) were used to calculate tbw ( l ) and ecw ( l ) using equations developed for use in older people ( mean age 67 , similar to our participants ) by vach and visser ( as quoted in ritz ( 22 ) ) , and tbw% , ecw% and icw% were calculated as percentages of body weight1 . total body water ( bw ) was calculated using vach equations ( 19 ) and used in estimating tbw : where r100 is impedance at 100hz , g is gender ( 0 for women and 1 for men ) and height and weight are measured in meters and kilograms . extracellular water was estimated using equations by visser ( as quoted in ritz ): where r5 is impedance at 5hz . tbw% , ecw% , icw% and ecw : icw ratio from the internal calculations of the mf - bia equipment , and those calculated from equations derived specifically for older people were each plotted in 22 tables against impending and current serum osmolality and calculated serum osmolarity . these tables were used to calculate sensitivity , specificity , positive predictive value ( ppv ) , negative predictive value ( npv ) , pre- and post - test probability of each for impending and current dehydration . where any of these values were not calculable due to the presence of zeros in the 22 table , 0.1 was added to each cell of the table . as published cut - off points of tbw , ecw and icw for dehydration are not readily available , three arbitrary cut - off points were selected for each measure ( tbw% , ecw% , icw% and the ratio ) . receiver operating characteristic ( roc ) curves were created for both impending and current dehydration , then additional promising cut - off points ( where cut - offs may possibly have both sensitivity and specificity > 60% ) were added to fill in the roc curves . at the ends of the roc curve , once either sensitivity or specificity was below 50% , no further outlying points were added . an acceptable cut - off point was considered to be one with both sensitivity and specificity greater than 60% and represented by the point closest to the top left corner of the roc plot . there is no definition of good enough sensitivity and specificity but we chose a minimum of 60% for both as suggesting that the measure was at least promising . for all cut - off points we also calculated positive predictive value ( ppv ) , negative predictive value ( npv ) , and positive and negative post - test probabilities . no sample size calculation was performed as there were no data available previously reporting diagnostic accuracy of mf - bia against serum osmolality . twenty seven participants were a realistic sample given the time frame we were able to use for this study . of the 27 included participants 12 ( 44% ) were well hydrated ( serum osmolality 275 to < 295 mosm / kg ) , 9 ( 33% ) had impending dehydration ( serum osmolality 295300 mosm / kg ) and 6 ( 22% ) were dehydrated ( serum osmolality > 300 mosm / kg ) . stratified by calculated serum osmolarity 8 ( 30% ) were well hydrated ( 275 to < 295 mosm / l ) , 7 ( 26% ) had impending dehydration , and 12 ( 44% ) had current dehydration ( > 300mosm / l ) ( table 1 ) . 11% ( n=3 ) were receiving a nil - by - mouth feeding regimen because of dysphagia ( as they were being fed nasogastrically ) . one patient was on pureed diet and 19% ( n=5 ) on soft - mashed diets due to slight swallowing difficulties . sixty seven percent ( n=18 ) were on normal oral diets without manipulation of food texture . serum osmolality , osmolarity , age , height and weight were normally distributed ( both kolmogorov - smirnov and wilk - shapiro p - values were > 0.05 suggesting that the data came from a normal distribution ) . cronbach s alpha ( ) was 0.960 for the reproducibility of the two impedance measures at 5 khz ( n=27 ) , suggesting excellent internal consistency . cronbach s alpha also suggested excellent internal consistency for impedance at 50 khz , and 100 khz , and tbw ( l ) ( 0.974 , 0.978 and 0.995 respectively ) . current dehydration ( > 300 mosm / l ) diagnosed on serum osmolarity criteria was twice as common as when based on serum osmolality ( > 300 mosm / kg ) , the reference standard ( table 2 ) . as calculated osmolarity ( in mosm / l ) is often used in clinical practice in place of measured osmolality ( in mosm / kg ) we directly compared the two for individuals . mosm / l while mean measured serum osmolality was 295.5 ( 7.5 ) mosm / kg . direct comparison showed that there was a significant difference of 2.72 ( 95% ci 0.6 to 4.8 ; p=0.014 ) . when stratified by hydration status serum osmolarity was greater than osmolality for hydrated participants ( mean difference 4.7 , 95% ci 1.1 to 8.2 , p=0.02 ) and those with impending dehydration ( mean difference 2.9 , 95% ci 0.2 to 5.6 , p=0.04 ) but not for those with current dehydration ( mean difference 1.4 , 95% ci 7.4 to 4.5 , p=0.57 ) . mean serum sodium , potassium , creatinine , urea and glucose values were always higher in those with current dehydration than those who were well hydrated ( table 2 ) , but the mean values for impending dehydration were not always between those of hydrated and currently dehydrated groups . there were few clear patterns in tbw% , ecw% , icw% or ecw : icw ratio by serum osmolality or calculated serum osmolarity ( table 2 ) . no cut - off point for tbw% , icw% , ecw% or ecw : icw ratio ( calculated by the mf - bia equipment ) had both a sensitivity and specificity above 60% for impending ( supplementary table 1 ) or current ( supplementary table 2 ) dehydration as assessed against ( measured ) serum osmolality . figure 1 shows the roc plot for icw% for impending dehydration by serum osmolality and figure 2 shows the roc plot for ecw% for current dehydration by serum osmolality ) . diagnostic accuracy for tbw% , icw% , ecw% and ecw : icw calculated using the equations specifically developed for older people ( rather than those programmed into the mf - bia equipment ) compared to serum osmolality resulted in no cut - off points with both sensitivity and specificity > 60% for impending dehydration ( supplementary table 3 ) , and one cut - off point for current dehydration ( supplementary table 4 ) . tbw% with a cut - off at 46% of body weight , was diagnostic of current dehydration by osmolality with sensitivity of 67% ( 95% ci 49% to 85% ) , specificity 62% ( 95% ci 44% to 80% ) ( supplementary table 4 , figure 3 ) . the positive likelihood ratio ( lr ) for this cut - off was 1.75 and negative likelihood ratio ( lr ) was 0.54 . no cut - off points for tbw% , icw% , ecw% or ecw : icw as calculated by the mf - bia equipment against calculated serum osmolarity had a sensitivity and specificity above 60% for impending ( 295 mosm / l serum osmolarity , supplementary table 5 ) or current dehydration ( 295 mosm / l , supplementary table 6 , suplementary figures 15 ) . diagnostic accuracy for water fractions calculated using the equations for older people against calculated serum osmolarity resulted in one cut - off point with both sensitivity and specificity of at least 60% . tbw% at 47% of body weight was diagnostic of impending dehydration by calculated osmolarity with sensitivity and specificity of 63% ( 95% ci 45% to 81% ) ( supplementary table 7 ; figure 4 ) . due to the very limited diagnostic accuracy of tbw% , icw% , ecw% or ecw : icw secondary analyses were considered . as the fluid content of fatty tissue is minimal we hypothesised that diagnostic accuracy might be improved if we separated men and women ( as men and women have different proportions of fatty tissue ) , or analysed total body water as a percentage of fat free mass ( lean body weight ) . analyses were run using total body water data as tbw% was the measure with most diagnostic accuracy in previous analyses . separating out men and women ( using tbw as a percentage of total body weight ) improved diagnostic accuracy for men for current dehydration ( using the ritz equations ) , and provided a cut - off for impending dehydration ( table 3 , supplementary tables 9 and 10 ) . the cut - off of 46% for tbw% showed sensitivity of 67% and specificity of 85% for current dehydration while 47% diagnosed impending dehydration ( sensitivity 63% , specificity 75% ) ( supplementary figures 6 and 7 ) . analysing tbw as a percentage of lean body weight provided a potentially useful cut - off for women for impending dehydration at 84% ( sensitivity 71% , specificity 75% ) , but none for current dehydration or for men alone , or men and women combined ( supplementary tables 11 and 12 , supplementary figures 810 ) . of the 27 included participants 12 ( 44% ) were well hydrated ( serum osmolality 275 to < 295 mosm / kg ) , 9 ( 33% ) had impending dehydration ( serum osmolality 295300 mosm / kg ) and 6 ( 22% ) were dehydrated ( serum osmolality > 300 mosm / kg ) . stratified by calculated serum osmolarity 8 ( 30% ) were well hydrated ( 275 to < 295 mosm / l ) , 7 ( 26% ) had impending dehydration , and 12 ( 44% ) had current dehydration ( > 300mosm / l ) ( table 1 ) . 11% ( n=3 ) were receiving a nil - by - mouth feeding regimen because of dysphagia ( as they were being fed nasogastrically ) . one patient was on pureed diet and 19% ( n=5 ) on soft - mashed diets due to slight swallowing difficulties . sixty seven percent ( n=18 ) were on normal oral diets without manipulation of food texture . serum osmolality , osmolarity , age , height and weight were normally distributed ( both kolmogorov - smirnov and wilk - shapiro p - values were > 0.05 suggesting that the data came from a normal distribution ) . cronbach s alpha ( ) was 0.960 for the reproducibility of the two impedance measures at 5 khz ( n=27 ) , suggesting excellent internal consistency . cronbach s alpha also suggested excellent internal consistency for impedance at 50 khz , and 100 khz , and tbw ( l ) ( 0.974 , 0.978 and 0.995 respectively ) . current dehydration ( > 300 mosm / l ) diagnosed on serum osmolarity criteria was twice as common as when based on serum osmolality ( > 300 mosm / kg ) , the reference standard ( table 2 ) . as calculated osmolarity ( in mosm / l ) is often used in clinical practice in place of measured osmolality ( in mosm / kg ) we directly compared the two for individuals . mosm / l while mean measured serum osmolality was 295.5 ( 7.5 ) mosm / kg . direct comparison showed that there was a significant difference of 2.72 ( 95% ci 0.6 to 4.8 ; p=0.014 ) . when stratified by hydration status serum osmolarity was greater than osmolality for hydrated participants ( mean difference 4.7 , 95% ci 1.1 to 8.2 , p=0.02 ) and those with impending dehydration ( mean difference 2.9 , 95% ci 0.2 to 5.6 , p=0.04 ) but not for those with current dehydration ( mean difference 1.4 , 95% ci 7.4 to 4.5 , p=0.57 ) . mean serum sodium , potassium , creatinine , urea and glucose values were always higher in those with current dehydration than those who were well hydrated ( table 2 ) , but the mean values for impending dehydration were not always between those of hydrated and currently dehydrated groups . there were few clear patterns in tbw% , ecw% , icw% or ecw : icw ratio by serum osmolality or calculated serum osmolarity ( table 2 ) . no cut - off point for tbw% , icw% , ecw% or ecw : icw ratio ( calculated by the mf - bia equipment ) had both a sensitivity and specificity above 60% for impending ( supplementary table 1 ) or current ( supplementary table 2 ) dehydration as assessed against ( measured ) serum osmolality . figure 1 shows the roc plot for icw% for impending dehydration by serum osmolality and figure 2 shows the roc plot for ecw% for current dehydration by serum osmolality ) . diagnostic accuracy for tbw% , icw% , ecw% and ecw : icw calculated using the equations specifically developed for older people ( rather than those programmed into the mf - bia equipment ) compared to serum osmolality resulted in no cut - off points with both sensitivity and specificity > 60% for impending dehydration ( supplementary table 3 ) , and one cut - off point for current dehydration ( supplementary table 4 ) . tbw% with a cut - off at 46% of body weight , was diagnostic of current dehydration by osmolality with sensitivity of 67% ( 95% ci 49% to 85% ) , specificity 62% ( 95% ci 44% to 80% ) ( supplementary table 4 , figure 3 ) . the positive likelihood ratio ( lr ) for this cut - off was 1.75 and no cut - off points for tbw% , icw% , ecw% or ecw : icw as calculated by the mf - bia equipment against calculated serum osmolarity had a sensitivity and specificity above 60% for impending ( 295 mosm / l serum osmolarity , supplementary table 5 ) or current dehydration ( 295 mosm / l , supplementary table 6 , suplementary figures 15 ) . diagnostic accuracy for water fractions calculated using the equations for older people against calculated serum osmolarity resulted in one cut - off point with both sensitivity and specificity of at least 60% . tbw% at 47% of body weight was diagnostic of impending dehydration by calculated osmolarity with sensitivity and specificity of 63% ( 95% ci 45% to 81% ) ( supplementary table 7 ; figure 4 ) . due to the very limited diagnostic accuracy of tbw% , icw% , ecw% or ecw : icw secondary analyses were considered . as the fluid content of fatty tissue is minimal we hypothesised that diagnostic accuracy might be improved if we separated men and women ( as men and women have different proportions of fatty tissue ) , or analysed total body water as a percentage of fat free mass ( lean body weight ) . analyses were run using total body water data as tbw% was the measure with most diagnostic accuracy in previous analyses . separating out men and women ( using tbw as a percentage of total body weight ) improved diagnostic accuracy for men for current dehydration ( using the ritz equations ) , and provided a cut - off for impending dehydration ( table 3 , supplementary tables 9 and 10 ) . the cut - off of 46% for tbw% showed sensitivity of 67% and specificity of 85% for current dehydration while 47% diagnosed impending dehydration ( sensitivity 63% , specificity 75% ) ( supplementary figures 6 and 7 ) . analysing tbw as a percentage of lean body weight provided a potentially useful cut - off for women for impending dehydration at 84% ( sensitivity 71% , specificity 75% ) , but none for current dehydration or for men alone , or men and women combined ( supplementary tables 11 and 12 , supplementary figures 810 ) . although we tried different ways of calculating tbw , icw and ecw , assessed tbw as a percentage of total body weight or lean body weight , separated participants by gender and defined dehydration using both serum osmolality and serum osmolarity only 8 cut - off points had both sensitivity and specificity of at least 60% . the only cut - off with limited diagnostic accuracy for both men and women was observed for tbw% at 46% when calculated using equations developed for older people ( sensitivity 67% , specificity 62% ) for current dehydration by measured osmolality ( > 300 mosmol / kg ) , but positive and negative likelihood ratios were poor ( 1.75 and 0.54 respectively ) . similarly tbw at 47% , only with equations developed for older people , also showed limited diagnostic accuracy ( sensitivity 63% and specificity 63% , lr 1.7 and lr 0.6 ) for impending dehydration as assessed by calculated serum osmolarity ( 295 mosmol / l ) . when internal equipment equations for estimating tbw were used no cut - offs were even minimally diagnostic . in secondary analyses cut - offs of tbw as a percentage of total body weight were partially useful in diagnosing impending and water - loss dehydration ( by serum osmolality ) in men , but none were useful in women . one cut - off for tbw as a percentage of lean body mass was useful in women in diagnosing impending dehydration , but none for current dehydration or in men . in this small population of 27 people with recent strokes , mf - bia did not fulfil its promise as a diagnostic tool for water - loss dehydration . calculated serum osmolarity was not good at predicting those with current dehydration by the reference standard , measured serum osmolality , and using calculated osmolarity resulted in 44% of our population being labelled as having current dehydration , compared to 22% by serum osmolality . research is needed to assess the health impacts of dehydration as measured by changes in serum osmolality , serum osmolarity and weight change , so that we can be sure which is the most useful measure of dehydration to use in future . the limited diagnostic accuracy for current dehydration by osmolality at tbw% of 46% ( sensitivity 67% , specificity 62% ) using the impedance output from mf - bia to calculate tbw% suggests that only 67 of every 100 people with current dehydration by serum osmolality will be positive using tbw% as the test , meaning that 33 of every 100 with current dehydration will be missed . similarly the specificity of 62% suggests that for every 100 people without current dehydration 62 will have a negative test but 38 will have a positive test2 . this is a very high level of false positives and negatives , suggesting that mf - bia is not useful in diagnosing water - loss dehydration independently of other clinical or biochemical data . the test s positive ( ppv ) and negative ( npv ) predictive values3 as well as pre and post - test probabilities provide more information on the utility of tbw% at the 46% cut off point . the ppv of 33% ( equivalent to the positive post - test probability of 33% ) suggests that only 33% of those who are diagnosed as having current dehydration by mf - bia truly have current dehydration by serum osmolality . the npv of 87% is clearly better , meaning that 87% of those diagnosed as not having current dehydration are truly without current dehydration ( and this is another way of stating the negative post - test probability of 13% ) . the positive likelihood ratio ( lr ) was 1.75 and negative likelihood ratio ( lr ) 0.544 suggesting that for a person positive for dehydration by this test the odds are 1.75 that dehydration is present compared to 1.00 for a person studies evaluating the utility of mf - bia in diagnosing dehydration in clinical settings are scarce . meta - analysis of studies in which tbw was estimated using bia and validated against the reference standard method , deuterium isotope dilution , found that mf - bia estimated tbw more closely to the reference standard ( wmd 0.18l , 95% ci 1.62 to 1.98 ) than either single frequency ( sf ) bia ( wmd 3.00l , 95% ci 1.43 to 4.57 ) or bioelectric impedance spectroscopy ( wmd 2.80l , 95% ci 1.03 to 4.58 ) . however , there are other indications in the literature that assessment of tbw may not be sufficiently accurate in conditions of change in hydration status and when body compartments are undergoing acute changes . this may be because changes in the ratio of intra- to extra - cellular water , and of acute changes in these compartments , also influence resistivity [ 2629 ] . this may mean that there are fundamental problems with mf - bia in assessing tbw and so in using mf - bia in predicting hydration status . our results suggesting that mf - bia is not a useful diagnostic tool are in broad agreement with those of olde rikkert 1997 . they found that in dehydrated geriatric patients ( n=53 ) the sensitivity of diagnosing dehydration using 100 khz mf - bia measurements was only 14% very poor sensitivity , and sensitivity was not improved when other frequencies were tested . the sensitivity and specificity of mf - bia were similar to that of the much simpler measure , tongue dryness , found in a recent australian study . tongue dryness is easier and quicker to assess , and does not need additional investment or electrical supplies , so would be a more appropriate measure to use in most situations than mf - bia if its diagnostic accuracy is similar . leaving the mathematics of diagnostic accuracy of mf - bia aside and observing data generated by mf - bia also suggested that mf - bia generated outcomes are not coherent with the diagnosis of dehydration . table 2 suggested no significant difference in mf - bia measures between hydrated , impending , and currently dehydrated groups . the intracellular water content reflects information on the state of hydration at the cellular level . cellular hydration status can change within minutes under the effects of stress , nutrients , hormones , and other factors . mf - bia does not appear to usefully reflect changes observed in serum osmolality or osmolarity or to sensitively identify the dehydrated state at the cellular level . haussinger suggested that while a well hydrated cell increases anabolic processes , a dehydrated cell shifts metabolism to catabolism , especially in muscle tissue . if recovery is to occur in a highly stressed patient after stroke , we want to ensure they are in an anabolic rather than a catabolic state . bhalla found that the 30% of their 167 stroke patients who had raised serum osmolality ( > 296 mosm / kg ) had increased odds of mortality at 3 months ( or 2.4 , 95%ci 1.0 t 5.9 ) . kelly found that in their 102 acute ischaemic stroke patients raised serum osmolality ( > 297 mosm / kg , in 24% of their patients ) on day 9 following admission was associated with increased odds of venous thromboembolism ( or 4.7 , 95% ci 1.4 to 16.3 ) . the maltron website states that the bioscan 920 - 2 multi - frequency analyser with its unique features is a rapid , non - invasive , inexpensive method for evaluating hydration and nutrition status it suggests applications in fluid retention and effects of hydration and dehydration . despite the maltron website reporting that it is quick , safe and easy and no assistance or technical knowledge is required ( see www.maltronint.com/popup_pages/bioscan9202.htm , accessed 23/2/2012 ) we found the machine is not user friendly . without a keyboard , data entry and saving of data are slow and may result in errors and data loss . re - running a second measurement for the same participant requires re - entering all the same information again or the new test overwrites existing data . analysed data are not easily accessible to visual check without downloading the full data set , and there is no warning when unrealistic readings are registered . on - site readout of each variable for each participant was time consuming and unrealistic in an acute stroke unit . in assessing mf - bia the battery was charged between measurements , cables were of appropriate length , and the equipment calibrated for all participants reported in this paper . weight and height were measured by the investigator or by a health professional ( not self - reported ) , mf - bia measurements were carried out at ambient temperature and manufacturer guidelines on positioning of electrodes were followed . before measurements our participants were in a supine position for least 10 min and during measurement they had no contact with any metal object ( such as a bed frame ) . electrodes were attached to the non affected side to record measurements and to skin with no abrasions or deformation that may affect current conductance . none of our participants suffered from edema due to excessive hydration by iv / electrolyte infusion or very low albumin levels . one of the main limitations in a ward setting is the inability of researchers to completely control participants fasting or bladder voidance , but the investigator did ensure that participants had fasted for at least 2 hours before mf - bia measurements were taken and all participants were asked if they would like to void their bladder before measurements commenced . study strengths included the use of serum osmolality and calculated osmolarity as reference standards , a population with high levels of dehydration , and recording serum osmolality and other serum measures ( sodium , potassium , glucose , urea ) within 20 minutes of mf - bia measurements ( enabling us to capture cellular hydration status as evaluated by mf - bia and its coherence with reference serum values ) . the limited diagnostic accuracy for current dehydration by osmolality at tbw% of 46% ( sensitivity 67% , specificity 62% ) using the impedance output from mf - bia to calculate tbw% suggests that only 67 of every 100 people with current dehydration by serum osmolality will be positive using tbw% as the test , meaning that 33 of every 100 with current dehydration will be missed . similarly the specificity of 62% suggests that for every 100 people without current dehydration 62 will have a negative test but 38 will have a positive test2 . this is a very high level of false positives and negatives , suggesting that mf - bia is not useful in diagnosing water - loss dehydration independently of other clinical or biochemical data . the test s positive ( ppv ) and negative ( npv ) predictive values3 as well as pre and post - test probabilities provide more information on the utility of tbw% at the 46% cut off point . the ppv of 33% ( equivalent to the positive post - test probability of 33% ) suggests that only 33% of those who are diagnosed as having current dehydration by mf - bia truly have current dehydration by serum osmolality . the npv of 87% is clearly better , meaning that 87% of those diagnosed as not having current dehydration are truly without current dehydration ( and this is another way of stating the negative post - test probability of 13% ) . the positive likelihood ratio ( lr ) was 1.75 and negative likelihood ratio ( lr ) 0.544 suggesting that for a person positive for dehydration by this test the odds are 1.75 that dehydration is present compared to 1.00 for a person studies evaluating the utility of mf - bia in diagnosing dehydration in clinical settings are scarce . meta - analysis of studies in which tbw was estimated using bia and validated against the reference standard method , deuterium isotope dilution , found that mf - bia estimated tbw more closely to the reference standard ( wmd 0.18l , 95% ci 1.62 to 1.98 ) than either single frequency ( sf ) bia ( wmd 3.00l , 95% ci 1.43 to 4.57 ) or bioelectric impedance spectroscopy ( wmd 2.80l , 95% ci 1.03 to 4.58 ) . however , there are other indications in the literature that assessment of tbw may not be sufficiently accurate in conditions of change in hydration status and when body compartments are undergoing acute changes . this may be because changes in the ratio of intra- to extra - cellular water , and of acute changes in these compartments , also influence resistivity [ 2629 ] . this may mean that there are fundamental problems with mf - bia in assessing tbw and so in using mf - bia in predicting hydration status . our results suggesting that mf - bia is not a useful diagnostic tool are in broad agreement with those of olde rikkert 1997 . they found that in dehydrated geriatric patients ( n=53 ) the sensitivity of diagnosing dehydration using 100 khz mf - bia measurements was only 14% very poor sensitivity , and sensitivity was not improved when other frequencies were tested . the sensitivity and specificity of mf - bia were similar to that of the much simpler measure , tongue dryness , found in a recent australian study . tongue dryness is easier and quicker to assess , and does not need additional investment or electrical supplies , so would be a more appropriate measure to use in most situations than mf - bia if its diagnostic accuracy is similar . leaving the mathematics of diagnostic accuracy of mf - bia aside and observing data generated by mf - bia also suggested that mf - bia generated outcomes are not coherent with the diagnosis of dehydration . table 2 suggested no significant difference in mf - bia measures between hydrated , impending , and currently dehydrated groups . . cellular hydration status can change within minutes under the effects of stress , nutrients , hormones , and other factors . mf - bia does not appear to usefully reflect changes observed in serum osmolality or osmolarity or to sensitively identify the dehydrated state at the cellular level . haussinger suggested that while a well hydrated cell increases anabolic processes , a dehydrated cell shifts metabolism to catabolism , especially in muscle tissue . if recovery is to occur in a highly stressed patient after stroke , we want to ensure they are in an anabolic rather than a catabolic state . bhalla found that the 30% of their 167 stroke patients who had raised serum osmolality ( > 296 mosm / kg ) had increased odds of mortality at 3 months ( or 2.4 , 95%ci 1.0 t 5.9 ) . kelly found that in their 102 acute ischaemic stroke patients raised serum osmolality ( > 297 mosm / kg , in 24% of their patients ) on day 9 following admission was associated with increased odds of venous thromboembolism ( or 4.7 , 95% ci 1.4 to 16.3 ) . the maltron website states that the bioscan 920 - 2 multi - frequency analyser with its unique features is a rapid , non - invasive , inexpensive method for evaluating hydration and nutrition status it suggests applications in fluid retention and effects of hydration and dehydration . despite the maltron website reporting that it is quick , safe and easy and no assistance or technical knowledge is required ( see www.maltronint.com/popup_pages/bioscan9202.htm , accessed 23/2/2012 ) we found the machine is not user friendly . without a keyboard , data entry and saving of data are slow and may result in errors and data loss . re - running a second measurement for the same participant requires re - entering all the same information again or the new test overwrites existing data . analysed data are not easily accessible to visual check without downloading the full data set , and there is no warning when unrealistic readings are registered . on - site readout of each variable for each participant was time consuming and unrealistic in an acute stroke unit . in assessing mf - bia we followed the guidelines of the european society of clinical nutrition and metabolism . the battery was charged between measurements , cables were of appropriate length , and the equipment calibrated for all participants reported in this paper . weight and height were measured by the investigator or by a health professional ( not self - reported ) , mf - bia measurements were carried out at ambient temperature and manufacturer guidelines on positioning of electrodes were followed . before measurements our participants were in a supine position for least 10 min and during measurement they had no contact with any metal object ( such as a bed frame ) . electrodes were attached to the non affected side to record measurements and to skin with no abrasions or deformation that may affect current conductance . none of our participants suffered from edema due to excessive hydration by iv / electrolyte infusion or very low albumin levels . one of the main limitations in a ward setting is the inability of researchers to completely control participants fasting or bladder voidance , but the investigator did ensure that participants had fasted for at least 2 hours before mf - bia measurements were taken and all participants were asked if they would like to void their bladder before measurements commenced . study strengths included the use of serum osmolality and calculated osmolarity as reference standards , a population with high levels of dehydration , and recording serum osmolality and other serum measures ( sodium , potassium , glucose , urea ) within 20 minutes of mf - bia measurements ( enabling us to capture cellular hydration status as evaluated by mf - bia and its coherence with reference serum values ) . mf - bia does not appear appropriate for the diagnosis of water - loss dehydration after stroke . diagnostic accuracy is far too low to usefully diagnose current or impending dehydration at any selected cut - off point . however , separating assessment by sex , and using tbw as a percentage of lean body weight may warrant further investigation .

backgroundnon - invasive methods for detecting water - loss dehydration following acute stroke would be clinically useful . we evaluated the diagnostic accuracy of multi - frequency bioelectrical impedance analysis ( mf - bia ) against reference standards serum osmolality and osmolarity.material/methodspatients admitted to an acute stroke unit were recruited . blood samples for electrolytes and osmolality were taken within 20 minutes of mf - bia . total body water ( tbw% ) , intracellular ( icw% ) and extracellular water ( ecw% ) , as percentages of total body weight , were calculated by mf - bia equipment and from impedance measures using published equations for older people . these were compared to hydration status ( based on serum osmolality and calculated osmolarity ) . the most promising receiver operating characteristics curves were plotted.results27 stroke patients were recruited ( mean age 71.3 , sd10.7 ) . only a tbw% cut - off at 46% was consistent with current dehydration ( serum osmolality > 300 mosm / kg ) and tbw% at 47% impending dehydration ( calculated osmolarity 295300 mosm / l ) with sensitivity and specificity both > 60% . even here diagnostic accuracy of mf - bia was poor , a third of those with dehydration were wrongly classified as hydrated and a third classified as dehydrated were well hydrated . secondary analyses assessing diagnostic accuracy of tbw% for men and women separately , and using tbw as a percentage of lean body mass showed some promise , but did not provide diagnostically accurate measures across the population.conclusionsmf-bia appears ineffective at diagnosing water - loss dehydration after stroke and can not be recommended as a test for dehydration , but separating assessment by sex , and using tbw as a percentage of lean body weight may warrant further investigation .

Background Material and Methods Statistical analysis Results Participant characteristics Internal consistency and reliability of MF-BIA measurements Dehydration status by osmolality and osmolarity Diagnostic accuracy of MF-BIA Diagnostic accuracy of MF-BIA Diagnostic accuracy of MF-BIA using TBW as a percentage of fat free mass Discussion Diagnostic accuracy The importance of MF-BIA results The convenience of the Maltron BioScan 920-2 Strengths and weaknesses Conclusions

PMC3800712

a 64-year - old woman came to our pain clinic with the symptom of right lateral side thigh pain which occurred from one year ago . her pain started from knee lateral side and the pain was radiated to buttock area . when she started to walk , the pain was triggered and whenever symptoms aggravated , she could feel the tingling sensation through a whole leg which made her limping . she described her pain as pin pricking or stabbing with her pain severitiy 7 - 8/10 on the visual analogue scale ( vas ) . before she felt the lateral leg pain however , we did not think it is the main problem causing the symptom . although her pain always started from knee lateral side , ultimately she could feel severe pain coming from entire thigh . therefore , we performed straight leg raising and patrick test to rule out any spine and pelvic problem . she was negative to all these tests and did not show any local tenderness around back or buttock . however , we could check significant local tenderness on right lateral side of knee . also we performed knee instability test on both side which showed no abnormality . as the pain always started from the knee and aggravated by walking , we suspected some pathologic changes from inside the knee like meniscus or cruciate ligament . we decided to evaluate knee magnetic resonance imaging ( mri ) study and simple x - ray . her mri study showed slight degenerative change on meniscus which seemed to be unrelated to the current pain . however , we could identify high signal intensity appearing at itb through coronal and axial image ( fig . 1 ) . we assessed that itb syndrome is causing the pain . to alleviate her symptom , we prescribed 37.5 mg tramadol/375 mg acetaminophen combination tablet ( paramacet tab : donga , seoul , korea ) , 5 mg amitriptyline per os twice daily but had little effect . we educated her about the itb specific stretching exercise which was known to improve the symptom by lengthening of the itb . she continued to perform this stretching exercise for a month but the pain relief was not satisfactory . to improve her symptom , we planned local corticosteroid injection by ultrasound guidance . we explained about the procedure , efficacy , and possible side effects of local corticosteroid injection . the skin was aseptically draped with betadine and the ultrasound transducer was covered with a sterile transparent sheath and aseptic gel . we used a 10 - 12 mhz linear transducer ( iu 22 , philips , netherlands ) . first , we marked the maximal tenderness area and the transducer was applied longitudinally to the lateral side of the knee . we could easily identify the itb , femoral lateral condyle , tibia , and recess . we targeted the needle tip beneath the iliotibial band and confirmed its location by injecting small amount of saline ( fig . after one week , the patient was followed up and she was fully recovered from previous pain . one month later , she was followed up again and she could not feel any pain around knee and thigh . itb syndrome causes pain in the area of the lateral femoral epicondyle or slightly below to it , which occurs after repetitive flexion and extension of the knee , typically in a runner , cyclist , or other athlete . an important finding of physical examination is local tenderness of the lateral knee below to the epicondyle and superior to the joint line . the noble test is performed with the patient lying supine ; beginning with the affected knee flexed at 90 , the leg is extended with direct pressure over the lateral femoral epicondyle , with reproducible pain near 30 of knee flexion . the ober test for distensibility of the itb is most commonly used to assess tightness of the itb . the thomas test is used to check the tightness of iliopsoas muscle , rectus femoris muscle , and itb . the differential diagnosis includes lateral meniscus tear , lateral compartment degenerative joint disease , biceps femoris tendinopathy , stress fracture , patellofemoral syndrome and lateral collateral ligament pathology . most of patients diagnosed as itb syndrome typically present symptoms localized to lateral side of the knee . however , the pain is not always localized to the lateral side of knee in which case our patient has shown , therefore , the diagnosis can be challenging in some cases . before she came to our pain clinic , she visited several other local clinics and has been heard that the thigh pain is coming from the spine disease like disc herniation or spinal stenosis . as the thigh pain is aggravated by walking or repetitive exercice , it can be considered as neurogenic intermittent claudication which is observed in spinal stenosis . the patients of itb syndrome can possibly complain thigh pain when we think the anatomical orientation of itb . the itb is formed proximally at the level of the greater trochanter as a coalescence of the tensor fascia latae and the gluteus maximus and medius muscles . anatomic reports have demonstrated that the itb is a dense fibrous connective tissue that passes distally along the thigh [ 7 - 9 ] . proximal to the knee joint , the itb has attachments to the intermuscular septum and the supracondylar tubercle of the femur . it continues inferiorly to insert on the gerdy tubercle at the anterolateral side of the tibia . the contact is formed at the level of the lateral femoral condyle between the itb and the underlying epicondyle and origin of the lateral collateral ligament , helping provide lateral stability to the knee joint . most of itb syndrome is a nontraumatic overuse injury which is caused by friction and rubbing of the distal portion of the itb over the lateral femoral epicondyle . orchard et al . proposed that an impingement zone occurs near 30 of knee flexion during foot strike and early stance phase . the itb passes over and posterior to the lateral femoral epicondyle at approximately 30 and greater of knee flexion . while this impingement happens , eccentric contraction of the tensor fascia lata and gluteus maximus is formed to exert great tension through the itb . the pathogenesis of itb syndrome involves inflammatory process and irritation of the posterior fibers of itb and lateral synovial recess . in cases of refractory itb syndrome itb syndrome can be diagnosed with the appearance of high - intensity signal on t2 weighted image which appears over the lateral epicondyle deep to the itb , and marked thickening of the distal itb . ultrasonography can also be used as a diagnostic tool and it demonstrates thickening of itb in case of patients . monitoring the thickness of itb might be a useful variable in the diagnosis of itb syndrome , moreover , to evaluate the evolution of this condition . oral nonsteroidal anti - inflammatory drugs and corticosteroid injections can be used to reduce the acute inflammatory response . gunter and schwellnus reported that local corticosteroid infiltration around itb ( 1 ml 1% lidocaine plus 40 mg methylprednisolone ) compared with control group effectively decreased pain during running in the first two weeks of treatment in patients with recent onset itb syndrome . . therefore , their report does not suggest any ideal location for corticosteroid injection . for symptom improvement of our patient the main benefit of ultrasound guidance during corticosteroid injection is the ability to identify vascular structures and nerves located in the needle pathway in order to avoid these structures and be sure to inject the medication into the most optimal location . ultrasonography enables real time examination , which promotes dynamic evaluation of various tendons , and continuous monitoring during any type of injection . in contrast to the fluoroscopy , ultrasound does not produce any harmful reaction to both physician and patient . moreover , ultrasound guided accurate injection can result in significantly greater clinical improvement in pain and function over blind ( landmark guided ) injections . for corticosteroid injection , initially , we identified several anatomical structure including the itb , lateral synovial recess and femoral epidcondyle . we targeted the potential space between the itb and overlying lateral femoral epicondyle because most of inflammatory and irritation process occurs here . the injection was successfully performed at this site without any event and the patient showed excellent recovery from pain . if the ultrasound guided corticosteroid injection of itb should be performed , we suggest that our method can be a useful option .

a 64-year - old woman visited our pain clinic with the pain of right lateral side of thigh for one year . her pain always started from knee and was radiated to buttock area when symptom was severe . she showed significant tenderness at knee lateral side and local tightness at lateral thigh . magnetic resonance image of the knee was performed and we could identify high signal intensity of iliotibial band through coronal and axial view . in spite of medication and physical stretching exercise of iliotibial band for one month , she did not show any improvement of pain . to alleviate her symptom , ultrasound guided local corticosteroid injection targeting beneath the iliotibial band was performed . after the procedure , the reduction of pain was significant and there was no need for further management .

CASE REPORT DISCUSSION

PMC4592921

keratoconus ( kc ) and fuchs ' dystrophy ( fuchs ) are the leading indications for penetrating keratoplasty ( pkp ) . the cornea surgeon 's main attention in corneal transplantation has shifted from preserving a clear graft to achieving a good refractive outcome . the nonmechanical excimer laser trephination ( exc ) has been first introduced in 1989 at the university eye hospital of erlangen ( germany ) [ 13 ] . it has been frequently reported that this technique yielded a better refractive outcome in comparison to manual trephination , particularly lower postoperative keratometric astigmatism , higher regularity of topography , and improved visual acuity [ 4 , 5 ] . it ensures an outstanding perpendicular incision profile . such cut edges in combination with orientation teeth ( figure 1 ) potentially reduce vertical tilt and horizontal torsion of the graft in the recipient bed , thus improving the visual performance after transplantation . however , despite the promising results , the exc pkp did not get widely spread , because corneal surgeons did not have an excimer laser in their operating theater . instead , newer technologies were introduced , particularly femtosecond laser - assisted keratoplasty ( flak ) , which got spread more widely since 2006 [ 68 ] . historically , the femtosecond laser has been mainly used in refractive surgery , for example , for flap preparation in lasik , intracorneal ring segment implantation in keratoconus patients , or antiastigmatic incisions following pkp [ 911 ] . the most common trephination profiles are the mushroom and the top hat profile as well as the more complex these shaped wound configurations offer the advantages of better donor - recipient fit and increased donor - host junction surface area contact , both resulting in faster wound healing and earlier suture removal , thus potentially promoting rapid visual recovery . in addition , in vivo confocal microscopy ( ivcm ) after flak showed earlier regrowth of corneal nerves in both the peripheral and central stroma compared to conventional pkp . to the best of our knowledge , this is the first study to compare two nonmechanical ( excimer and femtosecond laser ) laser - assisted pkp in keratoconus and fuchs dystrophy . the purpose of this work is to demonstrate the intraoperative results regarding the centration of the graft , necessity of completion of graft and donor incisions with scissors , anterior stepping , gaping , necessity of additional interrupted sutures , positive pressure during the surgery ( vis a tergo ) , and the proper alignment of the orientation markers in host and graft . in this prospective randomized clinical single - center study , 68 patients ( age 18 to 87 years ) with keratoconus or fuchs dystrophy ( phakic or pseudophakic eyes that underwent a primary central pkp ) were randomly distributed to 4 groups : 35 eyes with keratoconus and 33 eyes with fuchs dystrophy were treated either with excimer laser ( [ exc ] groups i and ii ) and with femtosecond laser - assisted ( [ flak ] groups iii and iv ) penetrating keratoplasty . exclusion criteria were repeated pkp and simultaneous cataract surgery because during the triple procedure , the iris - lens diaphragm is not stable and , therefore , might influence the main outcome measures of this study . all surgical procedures were carried out by one surgeon ( bs ) and under general anesthesia . in the study , a 193 nm excimer laser ( mel 70 , carl zeiss meditec , jena , germany ) with a 35 hz repetition rate and spot size of 1.2 mm , in combination with conventional donor / recipient masks , and the 60 khz intralase fs laser [ amo ( abbott medical optics ) , abbott park , il , usa ] have been used . exc and flak were performed in patients with keratoconus and with fuchs ' endothelial dystrophy ( if they presented advanced stages of the disease including scarring , thus they were not suitable for lamellar techniques such as dsaek or dmek ) . main intraoperative outcome measures included ultrasound pachymetry al-3000 ( tomey , nagoya , japan ) at the center and in 4 midperipheral points at 0 , 90 , 180 , and 270 of the donor , complications of laser trephination , trephination time , anterior gaping , graft override , need for additional interrupted sutures to achieve proper donor - host alignment , and alignment of orientation markers and positive vitreous pressure ( vis a tergo ) depicted in three grades ( 0 = no intraoperative pressure , 1 = iris prolapse till the level of the corneal incision , and 2 = iris prolapse beyond the level of corneal incision ) . graft decentration was measured at the end of the operation by measuring the distance between limbus and graft with calipers at the 12- and 6-o'clock position . decentration was considered if the difference between the two distances was more than 0.5 mm . a measurement prior to incision was not possible because of the suction ring for the femtolaser which prevents view on the limbus area . trephination was performed using the 193 nm excimer laser along metal masks with eight orientation teeth / notches . mean patient age in keratoconus was 37.4 14.2 and in fuchs 69.8 8.9 years . for donor trephination from the epithelial side using the 193 nm excimer laser mel 70 , a circular metal aperture mask ( diameter : 8.1 mm ; central opening : 3.0 mm for centration ; thickness : 0.5 mm ; weight : 0.173 g ; eight orientation teeth : 0.15 0.3 mm , figure 1 ) was positioned on a corneoscleral button ( 16 mm diameter ) fixed in an artificial anterior chamber under microscopic control ( figure 2 ) . after perforation , the remaining stromal lamellae and descemet 's membrane were cut with curved corneal microscissors . a corresponding circular metal mask was used ( diameter : 12.5 mm ; central opening : 8.0 mm ; thickness : 0.5 mm ; weight : 0.29 g ; eight orientation notches : 0.15 0.3 mm , figures 1 and 2 ) . the mask holds without additional stabilization because of the horizontal orientation of the patient 's head . the laser beam is guided automatically along the edge of the mask without ablating the central cornea . after focal corneal perforation , the remaining deep stromal lamellae and descemet 's membrane were cut with curved corneal microscissors . mean patient age in keratoconus group was 40.2 14.0 years and in the fuchs group it was 69.2 12.0 years . in all cases , we used energy of 0.1 j less than the maximum energy in the posterior side cut , 0.5 j less than the maximum energy in the anterior side cut , and 0.4 j less than the maximum energy in the ring lamellar cut ( 2.3 to 2.9 j ) . the 8 alignment incisions in both the donor and recipient were created as follows : energy of 1.5 j , length of 1000 m , width of 50 m , spot separation of 6 m , line separation of 6 m , and layer separation of 5 m . the radial offsets were + 2 in all recipients ( meaning that all the alignment incisions were outside the trephination ) and 2 in all donors ( meaning that all the alignment incisions were inside the trephination ) . on the anterior side cuts , the spot separation and the layer separation were 3 m ; in the ring lamellar cut ( spiral pattern ) , the tangential spot separation was 5 m and the radial spot separation was 4 m ; on the posterior side cut , the spot separation was 3 m and the layer separation was 2 m . the depth of the lamellar cut of the donor and recipient was 2/3 of the mean corneal thickness of the graft and recipient 's eye , respectively . all diameters ( anterior side cut , lamellar cut , and posterior side cut ) were performed , 0.1 mm larger than the resulting diameter , thus overlapping each other . the donor cornea was placed into an artificial anterior chamber type barron ( katena , denville , usa ) to achieve trephination from the epithelial side . each laser procedure requires a disposable glass interface , which applanates the cornea completely during the laser procedure . for laser trephination of the recipient 's cornea , the glass cone interface was placed within the suction ring so that the cornea was completely applanated . we performed a complete penetrating laser trephination after which the corneal button was removed with forceps and a spatula under microscopic control . if necessary , a microscissor was used to complete the incision . the top hat profile was used in fuchs dystrophy , whereas the mushroom profile was used in keratoconus patients ( figure 3 ) . in all patients , after temporary fixation of the donor button in the recipient bed with 8 interrupted sutures , a permanent wound closure was achieved by a 16-bite double - running diagonal cross - stitch suture ( 100 nylon ) according to hoffmann ( figure 4 ) . the eight cardinal sutures were placed at the site of orientation teeth with the excimer laser and at the site of the alignment incisions with the femtosecond laser as well as possible ( figure 5 ) . in cases of wound gaping or graft override , additional interrupted sutures were used to ensure proper donor - host alignment at the end of surgery . generally , the laser action time for trephination was much shorter for femtosecond compared to excimer laser trephination ( figure 6 ) . incisions had to be completed with scissors in almost all eyes of groups i / ii but only 2 cases in groups iii / iv . decentration happened in none of the eyes in groups i / ii , but in 3/1 eyes in groups iii / iv . no additional interrupted sutures were necessary for groups i / ii , but in 4/1 cases in groups iii / iv . orientation markers were aligned in all cases of the excimer groups ; in contrast , orientation markers were not totally aligned in 7/1 cases in groups iii / iv . after removal of 8 cardinal sutures , graft override appeared in none of groups i / ii / iv but in one case of group iii . moreover , gaping occurred in 0/1 eyes of groups i / ii but in 2/1 cases in groups iii / iv . intraoperative positive pressure from vitreous has occurred in all groups as follows : 3/9 eyes in groups i / ii and 8/11 in groups iii / iv ( figure 8) . in particular , positive vitreous pressure grade 2 appeared in 8 patients of the keratoconus flak group , but only in 1 patient of the fuchs excimer laser group . studies comparing an established corneal transplantation procedure with the new femtosecond laser technology that was introduced to clinical practice in 2006 were already carried out [ 18 , 19 ] . in a recent publication , birnbaum et al . have compared the results of 123 flak with conventional pkp in a randomized clinical study . it has been revealed that despite the potential advantages of the femtosecond procedure it did not provide superior refractive results as compared to mechanical trephination . they found a topographic astigmatism after suture removal of about 6 diopters in the flak group . a major advantage of femtosecond laser is the possibility to create different 3d profiles ( with the most widely spread ones being the top hat , mushroom , and zig - zag profile [ 13 , 18 ] ) . its stability is derived from the overlap , which is created by the side cut especially in the top hat configuration [ 20 , 21 ] . nevertheless , we found in our study that it was difficult to get it watertight without steps and gaps in comparison to the excimer laser keratoplasty . to avoid complications recorded in earlier studies using the mushroom profile ( e.g. , infiltrates , steps , and ointment deposits ) , we successfully used an anterior part of the mushroom as thick as two - thirds of the mean of midperipheral donor and recipient thickness . the fact that flak created more gaps after the removal of the cardinal sutures resulted in the necessity to use more single interrupted sutures for correct donor - host adaptation . this was not due to a learning curve of the surgeon because a different cut profile was used in the flak group . although , the mushroom profile has a relatively large diameter at the corneal surface , the wound apposition was less accurate . we have to admit that a suture depth of 90% can only be intended and depends strongly on the experience of the surgeon . we further have to admit that there are no ideal geometrical settings for the mushroom profile . but a more likely explanation is that in keratoconus eyes the applanation done to the cone - shaped bulging cornea results in flattening . we believe that this extreme flattening effect leads to an alteration in the theoretically planned right angles of the anterior , posterior , and lamellar side cuts . therefore , we got instead of right angles oblique angles and instead of a round trephination an oval- or pear - shaped trephination , which led to difficulty in fitting the properly cut donor button into the somehow distorted recipient bed . in particular , in keratoconus , orientation lines of donor and recipient tended not to match exactly ( figure 5 ) . in such flak cases , an interrupted suturing technique might be more appropriate than a double - running suture . it was obvious that the excimer laser needs more time to penetrate the cornea than the femtosecond laser . this is because the excimer laser digs from the surface a trench into the cornea while its energy is gradually absorbed . on the other hand , the femtosecond laser creates cavitation bubbles at different depths of the cornea and thus an incision can be obtained faster . we intended a complete perforation of the cornea during flak . if the femtosecond laser theatre is separated from the surgery room , it might be preferable to leave a stromal gap between 50 and 80 m and to complete perforation in the surgery room . flak achieved an overwhelming number of cases with complete perforation of the cornea in comparison to the excimer pkp . the double continuous running suture technique remains to be the suture of choice in flak . in our study , the suturing was done down deep to the pre - descemet 's layer and not just at the level of the side cut of the profiled graft . in an ordinary pkp or exc pkp , the suture is placed in the pre - descemet 's region of the donor cornea . in germany , the double - running cross - stitch suture according to hoffmann is preferred over interrupted sutures , because it results in earlier visual rehabilitation and higher regularity of topography as long as the sutures are in place and a lower risk of suture loosening and need of suture replacement . in case of corneal thinning , such as that in keratoconus , the suture may run through the anterior chamber at the recipient site . one of the disadvantages of flak is that it generates a higher intraocular pressure than the exc pkp . therefore , an increase in the volume of choroidal blood vessels produces disproportionate changes of intraocular volume and thus intraocular pressure . moreover , it leads to major changes in the osmolarity of the vitreous . when the cornea is removed , the mechanical barrier to vitreous expansion is lost . in this situation , the iris - lens diaphragm is pushed forward ( the so - called positive vitreous pressure or vis a tergo ) . in severe cases , it is impossible to maintain the anterior chamber and the cardinal sutures are difficult to place . even the iris can be sutured to the corneal button thus leading to further difficulties . moreover , a sudden increase in the intraocular pressure to values which are higher than the perfusion of the retinal vessels was recorded . however , this effect is only for a short time , due to the fast perforation of the cornea . up to now , no central artery occlusion due to flak has been reported . by leaving a stromal gap and finishing the perforation in the surgery room this leads to reduction of pressure on the eye , thus enabling intraocular pressure reaching equilibrium and reducing positive vitreous pressure . moreover , there are now also faster femtosecond laser platforms available which may help to further minimize this risk . decentration of corneal grafts in keratoconus patients with flak was more frequent than that in patients that were treated with excimer laser . because of the flat applanation with the flak , it was more likely to obtain a decentered oval - shaped recipient incision . in contrast , in the excimer laser pkp , we have been using masks with orientation teeth which allow us to suture the first eight cardinal sutures with small risk of horizontal torsion because , according to the key - keyhole - principle , the orientation teeth in the donor fit exactly into the orientation notches of the recipient . such a precise orientation is absent in the flak , since only radial markings are present . it became obvious that in the femtosecond laser trephined kc eyes these radial incision lines did not fit completely comparing donor and recipient . therefore , the hypothesis that a better graft alignment can be achieved with flak can not be confirmed with the geometrical settings in our study . intraoperative decentration , misalignment of the donor in the recipient bed , and need for additional interrupted sutures , as well as positive pressure from the vitreous , were more frequent when flak was performed . future comparative studies with a faster excimer laser platform and a concave femtosecond laser patient interface or even a liquid interface are needed to be carried out . the next step of our group is the presentation of best - spectacle corrected visual acuity , postoperative astigmatism , and regularity of topography after removal of all sutures in all eyes .

purpose . to assess the intraoperative results comparing two non - mechanical laser assisted penetrating keratoplasty approaches in keratoconus and fuchs dystrophy . patients and methods . 68 patients ( age 18 to 87 years ) with keratoconus or fuchs dystrophy were randomly distributed to 4 groups . 35 eyes with keratoconus and 33 eyes with fuchs dystrophy were treated with either excimer laser ( [ exc ] groups i and ii ) or femtosecond laser - assisted ( [ flak ] groups iii and iv ) penetrating keratoplasty . main intraoperative outcome measures included intraoperative decentration , need for additional interrupted sutures , alignment of orientation markers , and intraocular positive pressure ( vis a tergo ) . results . intraoperative recipient decentration occurred in 4 eyes of groups iii / iv but in none of groups i / ii . additional interrupted sutures were not necessary in groups i / ii but in 5 eyes of groups iii / iv . orientation markers were all aligned in groups i / ii but were partly misaligned in 8 eyes of groups iii / iv . intraocular positive pressure grade was recognized in 12 eyes of groups i / ii and in 19 eyes of groups iii / iv . in particular , in group iii , severe vis a tergo occurred in 8 eyes . conclusions . intraoperative decentration , misalignment of the donor in the recipient bed , and need for additional interrupted sutures as well as high percentage of severe intraocular positive pressure were predominantly present in the femtosecond laser in keratoconus eyes .

1. Introduction 2. Patients and Methods 3. Results 4. Discussion 5. Conclusion

PMC2897044

due to its distinctive layer structure and electronic properties , the inorganic fullerene - like structure of ms2(m = mo , w ) has been attracting considerable attention and has been investigated extensively for various applications . the properties of the metal disulfide nanomaterials with novel morphology are better than those of their bulk forms . ws2 and mos2 nanomaterials have shown excellent properties and have potential applications in various fields , such as scanning probe microscopy , solid - state lubricants , heterogeneous catalysis , and electrochemical hydrogen storage . in order to investigate their properties and applications in these fields systematically , several methods have been reported to prepare nanoscale ms2 , such as gas solid or gas phase reaction , pulsed laser deposition , a self - transformation process , and sulfidation of the moo3 nanofibers under a h2s / h2 steam at 350600c . however , almost all the methods are based on utilizing high temperature reactions or dangerous gases , such as h2 and h2s . on the other hand , the formation of nanosized mos2 or ws2 polyhedral or onion crystals is , similar to the case of carbon onion crystals , a rare event . from this point of view , it is interesting and valuable to find a new large - scale approach to synthesize metal disulfide nanoparticles . since the 1970s , lamellar disulfide has been intensively studied , especially via the intercalation technique [ 10 - 12 ] . during intercalation , the coordination patterns of lamellar disulfide change greatly , and numerous defects are introduced , which enable doping of other ions . on the other hand , after transition metal cations have entered the ws2 or mos2 matrix , there are changes in the bonding properties of the matrix and the s m golub et al . described room temperature preparation of new ternary metal sulfides mxmos2 ( m = cu , ag ) by exfoliation first , the mos2 powder is exfoliated into lamellae or monolayers by n - butyllithium to form lixmos2 . they supposed that ternary metal sulfides mxmos2 could be obtained using ( mos2 ) single layers dispersed in aqueous media and salts of metals which could favor the formation of m s rather than m thus , the ( mos2 ) single layers in the dispersions have henceforth to be considered not only as macroanions but also as reducing agents . for the mos2 , the negative charge of ( mos2 ) and consequently the cluster structure of s mo mo metal bonding can be effectively stabilized in restacked compounds by introduction into the structure of counterions covalently bonded to the matrix . so it is possible the same reason for the ws2 , the introduction of cobalt ion into the matrix of ws2 can stabilize the ws2 . utilized these bond changes and fabricated transition metal - doped disulfide nanotubes successfully by exfoliation intercalation through hydrothermal adulteration , using lamellar metal disulfide as the precursors . ws2 and mos2 could be electrode materials for rechargeable batteries due to the existence of van der waals forces across the gaps between the s m s sheets , which can provide the space for guest reactants in intercalation reactions . a few attempts have been made to investigate the electrochemical activity of ms2 ( m = mo , w , etc . ) for example , feng reported that ws2 nanoflakes prepared by a rheological phase reaction had better lithium intercalation / deintercalation behavior than other forms of ws2 . wang prepared ws2 nanotubes and reported that the ws2 nanotube electrode delivered a lithium insertion capacity of 915 mah g , which is much higher than for ws2 powder . as other approach to explore new electrode materials for li - battery , nanosized transition metal oxides have been attracting much attention in recent years . reported the high performance as anode materials in lithium ion batteries for nanosized transition metal oxides such as nickel oxide , cobalt oxide , and iron oxide . among them , cobalt oxide demonstrated the best electrochemical properties . in this regard , co - containing species can be promising intercalants to modify the lamellar disulfides . in this paper , bamboo - like co - doped ws2 nanorods were synthesized on a large scale by an approach involving exfoliation intercalation hydrothermal treatment , using lamellar tungsten disulfide as the precursor , and the morphology , chemical components , and raman spectrum are reported . the cobalt - doped ws2 nanorods can reversibly store lithium with a capacity of 568 mah g over a voltage range of 0.013.0 v versus li / li and show good cycling stability . to the best of our knowledge , this is the first time that transition metal - doped ws2 nanorods have been synthesized and their applications in lithium ion batteries systematically investigated . under n2 atmosphere at room temperature for a week , 8.2 g of ws2 powder was exfoliated by 2.9 m of n - butyllithium ( 15 ml ) in a container , rinsed in hexane six times , and vacuum dried at 80c overnight . then , 0.6 g of lixws2 was put into a 50 ml autoclave with a teflon - lined inner wall , and 30 ml of 0.2 m cocl2 aqueous solution was added into the autoclave and stirred for 15 min . the temperature of the autoclave was raised to 180c , and it was maintained at that temperature for 48 h before being cooled down to room temperature . the products were filtrated , rinsed in deionized water several times , and dried at 80c overnight . x - ray diffraction ( xrd ) was performed with a rigaku x - ray diffractometer at room temperature , using a quartz monochromator cu k radiation source ( = 0.1541 nm ) under a voltage of 40 kv and a current of 30 ma . transmission electron microscopy ( tem ) was performed with a fei / tecnai g microscope . before characterization , the samples were dispersed ultrasonically in ethanol and dropped onto a cu grid coated with a carbon membrane . energy - dispersive x - ray spectroscopy ( edx ) was performed on an s-4800 scanning electron microscope . raman spectra were collected under ambient conditions using a rm 2000 microscope confocal raman spectrometer equipped with a microscope ( renishaw plc , england ) . electrochemical measurements were carried out using a 2032-type coin cell fabricated in an argon - filled glove box ( mbraun , germany ) . the working electrode was fabricated in the ratio of 70:20:10 ( w / w ) active material / carbon black / polyvinylidene difluoride , while lithium foil served as counter and reference electrode . the electrolyte was 1 m lipf6 dissolved in 1:1 ethylene carbonate ( ec ) and dimethyl carbonate ( dmc ) . constant current charge / discharge cycling was conducted on a land battery tester ( ct2001a ) in the voltage range between 3.0 and 0.01 v. cyclic voltammetry ( cv ) was performed on a chi660c electrochemical workstation at a scan rate of 0.1 mv s. under n2 atmosphere at room temperature for a week , 8.2 g of ws2 powder was exfoliated by 2.9 m of n - butyllithium ( 15 ml ) in a container , rinsed in hexane six times , and vacuum dried at 80c overnight . then , 0.6 g of lixws2 was put into a 50 ml autoclave with a teflon - lined inner wall , and 30 ml of 0.2 m cocl2 aqueous solution was added into the autoclave and stirred for 15 min . the temperature of the autoclave was raised to 180c , and it was maintained at that temperature for 48 h before being cooled down to room temperature . the products were filtrated , rinsed in deionized water several times , and dried at 80c overnight . x - ray diffraction ( xrd ) was performed with a rigaku x - ray diffractometer at room temperature , using a quartz monochromator cu k radiation source ( = 0.1541 nm ) under a voltage of 40 kv and a current of 30 ma . transmission electron microscopy ( tem ) was performed with a fei / tecnai g microscope . before characterization , the samples were dispersed ultrasonically in ethanol and dropped onto a cu grid coated with a carbon membrane . energy - dispersive x - ray spectroscopy ( edx ) was performed on an s-4800 scanning electron microscope . raman spectra were collected under ambient conditions using a rm 2000 microscope confocal raman spectrometer equipped with a microscope ( renishaw plc , england ) . electrochemical measurements were carried out using a 2032-type coin cell fabricated in an argon - filled glove box ( mbraun , germany ) . the working electrode was fabricated in the ratio of 70:20:10 ( w / w ) active material / carbon black / polyvinylidene difluoride , while lithium foil served as counter and reference electrode . the electrolyte was 1 m lipf6 dissolved in 1:1 ethylene carbonate ( ec ) and dimethyl carbonate ( dmc ) . constant current charge / discharge cycling was conducted on a land battery tester ( ct2001a ) in the voltage range between 3.0 and 0.01 v. cyclic voltammetry ( cv ) was performed on a chi660c electrochemical workstation at a scan rate of 0.1 mv s. figure 1 shows the xrd patterns of the raw ws2 powder and the co - doped ws2 nanorods . all the peaks are agreement with the bragg positions for a hexagonal structure ( jcpds card 08 - 0237 ) for both samples . the co - doped ws2 nanorods , however , show significantly broadened peak shapes , indicating that the crystallite size is much smaller than that of the raw ws2 powder . the edx analysis ( fig . 2 ) indicates that these nanorods have a co : w : s atomic ratio of 3.54:31.4:58.03 . taking into account the error of the measurement , xrd patterns of the raw ws2 powder ( a ) and the co - doped ws2 sample ( b ) edx results on co - doped ws2 nanorods . the silicon peak comes from silicon wafer supporting the sample figure 3a shows a tem image of the raw ws2 particles . the selected area electron diffraction ( saed ) pattern reveals that the raw ws2 consists of single crystals of 2h - ws2 . as shown in fig . 3b , the morphology of the co - doped ws2 particles is that of bamboo - like nanorods with a diameter of about 20 nm and a length of around 200 nm , so the aspect ratio of these nanorods is about 10 . the corresponding saed pattern reveals that the nanorods are polycrystalline 2h - ws2 , which is different from the raw ws2 particles . the nanorods have somewhat irregular shape , in which the ends of a nanorod are smaller in diameter than its middle part , as shown in fig . tem images of the raw ws2 ( a ) , the co - doped ws2 ( b ) , and the co - doped ws2 at higher magnification ( c , d ) . the insets in a and b show the corresponding saed patterns the raman spectrum of the sample was collected using a he / ne laser with a wavelength of 632.8 nm , which is capable of supplying power of 1 mw . the raman spectrum of the co - doped ws2 nanorods displays two peaks around 348.6 and 419.6 cm , and no other additional peaks are observed , as shown in fig . this result is similar to that for ws2 bulk material and is consistent with reference , indicating the local lattice structure of the co - doped nanorods is comparable with that of the bulk ws2 crystals in spite of its poor crystallinity . raman spectrum of co - doped ws2 nanorods the electrochemical properties of the co - doped ws2 nanorods were compared with those of the raw ws2 powder . figure 5a , 5b show cyclic voltammograms for the two samples . in the first cycle , a reduction peak is observed at ~0.55 v and an oxidation peak at ~2.4 v for both samples . the reduction and the oxidation peaks correspond to li lithium intercalation / deintercalation behavior in ws2 for two samples , respectively , as described in eq . . cyclic voltammograms of a coin cell ( vs. li ) for selected cycles of the raw ws2 electrode ( a ) , the co - doped ws2 electrode ( b ) , and the co - doped ws2 at different potential scan rates ( v s ) ( c ) , ( d ) show dependence of the peak height on the square root of the potential scan rate for the oxidation peak in the second cycle , the reduction peak at 0.55 v has disappeared , while new reduction peaks appear in the potential range from 1.62.2 v. this change can be explained by the formation of a gel - like polymeric layer . there was no significant change in the potentials of the redox peaks afterward for co - doped ws2 . the main difference between two cyclic voltammograms is the relative change in the redox current with the cycle number ; the current of the redox peak for the co - doped ws2 electrode decreases more slowly than that of the raw ws2 electrode . as a result , a better cycling performance can be obtained from the co - doped ws2 electrode , as shown in fig . 6a . typical first and second charge / discharge curves of the co - doped ws2 electrode ( a ) , typical cycling performance of the precursor ws2 and the as - prepared co - doped ws2 electrode ( b ) and coulombic efficiency versus cycle number of the raw and co - doped ws2 ( c ) . current density : 50 ma g figure 5c shows cyclic voltammograms of the co - doped ws2 at potential scan rates , , from 20 to 500 v s. the relationship between the oxidation current peak values and is depicted in fig . the peak currents are proportional to , which may indicate a semi - infinite diffusion mechanism on the electrode . the relationship to peak current ip can be expressed by the classical sevcik equation : where n is the number of electrons per species reaction , a is the apparent surface area of the electrode , d0 is the diffusion coefficient of li in the solid state , is the scan rate , and c0 is the change in li concentration in the active material . figure 6a shows the charge / discharge curves of the electrode containing the co - doped ws2 nanorods . in the first cycle , the ws2 electrode delivered a lithium insertion capacity of about 668 mah g when the discharge / charge current density was 50 ma g. the first discharge curve shows the insertion plateaus at 1.0 v and 0.75 v. in the second cycle , a lithium insertion plateau at ~2.2 v was observed , as well as a slope starting from 1.75 v and running down to the cut off voltage of 0.01 v. there is a clear distinction in the charge discharge profile between 02 v ( ~300 mah g ) and 23 v ( ~250 mah g ) . thus , this material can be regarded as a possible anode material for li - ion battery in the 02 v range and as a cathode material in li - metal cell technology . the charge capacity obviously decreases with cycling , from 520 mah g for the first cycle to 287 mah g at the 40th cycle . ( the capacity retention is ~55% after 40 cycles . ) for the co - doped ws2 electrode , an irreversible capacity of 668 mah g is observed in the first cycle , along with a reversible capacity of 568 mah g , which is reduced to 446 mah g after 20 cycles and 380 mah g after 40 cycles . the co - doped ws2 electrode shows a better cycling stability compared with the ws2 precursor electrode . the first cycle coulombic efficiency of the raw ws2 is 81.5% , and it increases with the cycle number , reaching 98.2% by the 40th cycle . for the co - doped ws2 , the coulombic efficiency of the first cycle is 84.7% and over 98% from the 4th cycle , shown in fig . the improved electrochemical performance of the co - doped ws2 sample may be attributed to the small particle size and its one - dimensional structure . however , the charge / discharge capacities of the co - doped ws2 electrode are lower than those reported for ws2 nanoflakes and ws2 nanotubes . the formation mechanism for the nanorods is similar to the synthesis of transition metal - doped moxw1xs2 nanotubes . first , the ws2 powder is exfoliated into lamellae or monolayers by n - butyllithium to form lixws2 , with the process introducing a large amount of defects , along with large changes in the coordination patterns of the lamellar disulfide , which enables doping with other ions . then , the cocl2 aqueous solution is added into the above mixture , a dispersion of single - layer ws2 is formed by the reaction of lixws2 with water . after co cations enter into the ws2 matrix , the bonding properties of the matrix and the bond angles of s w s are changed , especially on the sides of layers , which causes the lamellar structure to curl into bamboo - like nanorods . the decreased capacity of this transition metal - doped ws2 electrode may be because the intercalation of transition metal ions into the ws2 matrix can decrease the distance between layers , which limits the amount of lithium ions electrochemically inserted into the co - doped ws2 . based on previous research [ 19 - 21 ] , lithium ions intercalate into ws2 nanoclusters and defect sites in ws2 nanorods , but these sites are partly saturated with co , so the amount of intercalation of lithium ions will be decreased , and the capacity could thus also be decreased . it can be deduced that for ws2 samples without transition metal - doping or mesoporous ws2 , this exfoliation restacking method will both enhance the charge / discharge capacity and achieve better ws2 cycling stability . further work is needed in order to further improve the electrochemical performance of ws2 samples via exfoliation and restacking , as well as other modifications . bamboo - like co - doped ws2 nanorods were synthesized by an exfoliation intercalation hydrothermal method . ws2 co cations were doped into the ws2 matrix through a hydrothermal process in cocl2 aqueous solution . this method is expected to be applied to other layered materials to obtain the desired morphologies . the co - doped ws2 nanorods can reversibly store lithium with a capacity of 568 mah g over a voltage range of 0.013.0 v versus li / li . the co - doped ws2 electrode shows much better cycling stability compared with the raw ws2 electrode . this work was supported partially by the state key laboratory breeding base of green chemistry - synthesis technology , zhejiang university of technology and the priority research centers program through the national research foundation of korea ( nrf ) funded by the ministry of education , science and technology ( 2009 - 0094047 ) . this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited . this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited .

cobalt - doped tungsten disulfide nanorods were synthesized by an approach involving exfoliation , intercalation , and the hydrothermal process , using commercial ws2 powder as the precursor and n - butyllithium as the exfoliating reagent . xrd results indicate that the crystal phase of the sample is 2h - ws2 . tem images show that the sample consists of bamboo - like nanorods with a diameter of around 20 nm and a length of about 200 nm . the co - doped ws2 nanorods exhibit the reversible capacity of 568 mah g1 in a voltage range of 0.013.0 v versus li / li+ . as an electrode material for the lithium battery , the co - doped ws2 nanorods show enhanced charge capacity and cycling stability compared with the raw ws2 powder .

Introduction Experimental Sample Preparation Characterization Results and Discussions Conclusions Acknowledgments Open Access

PMC4028924

cobb 's collar or moormann 's ring is a largely underestimated cause of male bulbar urethral strictures . it can potentially lead to acute urinary retention , upper urinary tract dilatations , enuresis , infection , poor streaming , and hematuria . embryologically , this focal bulbar urethral stricture develops due to failure of canalization of the cloacal membrane during fetal development . in cobb 's collar , the site of obstruction is more distal than that seen with posterior urethral valves ( puv ) . a prompt diagnosis with minimally invasive treatment may avoid upper and lower tract deterioration in these patients . we present two cases of congenital bulbar urethral obstruction ( cobb 's collar ) occurring in brothers . the anatomy , embryology , and clinical features are discussed in order to turn the spotlight on to this rare disease . a 30-year - old man presented with a long history of a worsening urinary stream and a sense of incomplete voiding and straining at micturition . there was no history of traumatic injury , instrumentation of the urethra , or sexually transmitted disease . abdominal and genitourinary examination revealed an enlarged and distended urinary bladder with normal - looking external genitalia . renal function tests and urine routine examination were within normal limits , and urine culture showed no growth . usg of the abdomen revealed bilateral mild hydronephrosis [ figure 1a ] , presence of hydroureters , a dilated prostatic urethra , and a thick - walled over - distended bladder . pre - void ( 1972 ml ) , first ( 957 ml ) and second post - void ( 790 ml ) residual volumes were significantly raised [ figure 1b ] . urodynamic study could not be performed as catheterization failed due to the tightness of the urethral stricture . subsequent retrograde urethrogram ( rgu ) showed a 3-cm stricture in the bulbar urethra [ figure 2a ] . micturating cystourethrogram ( mcu ) using the suprapubic route showed a narrowing in the proximal bulbar urethra , without reflux into the collecting system [ figure 2b ] . suprapubic catheterization ( spc ) with visual internal urethrotomy ( viu ) was performed and the patient was discharged . later , he was readmitted for a voiding trial and teflon dilatation . in view of the persistent urinary problems , the postoperative course was uneventful , and the patient voids without complaints at 6 months follow - up . ( a ) bilateral mild hydronephrosis ( b ) significant post - void urine residue ( case 1 ) ( a ) retrograde urethrogram demonstrating a narrowing in the proximal bulbar urethra , ( b ) micturating cystourethrogram ( mcu ) showed a narrowing in the proximal bulbar urethra ( white ring ) ( case 1 ) the first case 's 25-year - old younger brother also complained of poor urinary stream and straining at micturition . usg showed mild hydronephrotic changes , hydroureters , and a thickened bladder wall , with significant post - voiding residue . rgu was performed which showed a short segment stricture in the bulbar urethra [ figure 3 ] . the patient is asymptomatic at 6 months follow - up , with normal serial usg evaluations . a 30-year - old man presented with a long history of a worsening urinary stream and a sense of incomplete voiding and straining at micturition . there was no history of traumatic injury , instrumentation of the urethra , or sexually transmitted disease . abdominal and genitourinary examination revealed an enlarged and distended urinary bladder with normal - looking external genitalia . renal function tests and urine routine examination were within normal limits , and urine culture showed no growth . usg of the abdomen revealed bilateral mild hydronephrosis [ figure 1a ] , presence of hydroureters , a dilated prostatic urethra , and a thick - walled over - distended bladder . pre - void ( 1972 ml ) , first ( 957 ml ) and second post - void ( 790 ml ) residual volumes were significantly raised [ figure 1b ] . urodynamic study could not be performed as catheterization failed due to the tightness of the urethral stricture . subsequent retrograde urethrogram ( rgu ) showed a 3-cm stricture in the bulbar urethra [ figure 2a ] . micturating cystourethrogram ( mcu ) using the suprapubic route showed a narrowing in the proximal bulbar urethra , without reflux into the collecting system [ figure 2b ] . suprapubic catheterization ( spc ) with visual internal urethrotomy ( viu ) was performed and the patient was discharged . later , he was readmitted for a voiding trial and teflon dilatation . in view of the persistent urinary problems , the postoperative course was uneventful , and the patient voids without complaints at 6 months follow - up . ( a ) bilateral mild hydronephrosis ( b ) significant post - void urine residue ( case 1 ) ( a ) retrograde urethrogram demonstrating a narrowing in the proximal bulbar urethra , ( b ) micturating cystourethrogram ( mcu ) showed a narrowing in the proximal bulbar urethra ( white ring ) ( case 1 ) the first case 's 25-year - old younger brother also complained of poor urinary stream and straining at micturition . usg showed mild hydronephrotic changes , hydroureters , and a thickened bladder wall , with significant post - voiding residue . rgu was performed which showed a short segment stricture in the bulbar urethra [ figure 3 ] . the patient is asymptomatic at 6 months follow - up , with normal serial usg evaluations . the proximal prostatic and membranous part of the male urethra is formed by endodermal tissues and the penile part is formed from the phallic portion of the urogenital sinus . congenital idiopathic urethral stricture may result from incomplete rupture of the cloacal membrane at the junction of membranous and bulbar urethra which may correspond to submeatal stenosis in girls . 1968 ) were the first to draw attention to the entity of congenital stricture of the bulbar urethra . they analyzed 52 cases of proximal bulbar urethral obstruction in male patients for a 3-year period . twenty - six out of 52 patients were boys under 16 years of age ; of these 26 boys , 16 had a trabeculated bladder , 15 showed a dilated prostatic fossa , whereas 6 had a secondary hydroureter , and the most frequently referred symptoms were enuresis , urinary tract infection , hematuria , and failure to thrive . all these forms of obstruction are located just below the external sphincter [ figure 4 ] . it can appear either as a ridge of tissue ( cobb 's collar i ) or as a definite stricture in the area described ( cobb 's collar ii ) or even as a very tight pinhole opening ( cobb 's collar iii ) . p , prostate ; s , external sphincter ; c , cobb 's collar such strictures of the bulbar urethra had been underestimated for many years until the first half of the 1990s when dewan et al . and later nonomura et al . redefined and classified the bulbar urethral narrowing by reviewing video - recorded cystoscopies as well as radiological pictures in the patients . both studies showed that the majority of patients were below 1 year of age with a large number showing vesicoureteral reflux ( vur ) , with no history of urethral trauma , catheterization , or instrumentation . vur in association with clinical findings and endoscopic findings of vestigial remnants at the level of bulbar urethra indicates that this narrowing has a congenital onset . presentation at a later age , as occurred in our cases , may represent delayed presentation of a congenital stricture . their response to treatment is clearly different from that of the under 1 year olds , since majority require buccal mucosal onlay urethroplasty or scrotal or penile island flap urethroplasty . the more common causes of congenital urethral stricture are different types of posterior urethral obstruction , which was originally described as puv ( three types ) by young et al . , but these obstructions were found to be diaphragmatic obstruction as suggested by parkkulainen on endoscopic studies . congenital attachment of the verumontanum to the anterior wall of the posterior urethra is the embryologic forerunner to the congenital obstruction of the posterior urethra . congenital obstructing posterior urethral membrane ( copum ) , first described by dewan et al . more distal bulbar urethral abnormality without folds running up to the verumontanum is likely to be a remnant of urogenital diaphragm , commonly known as cobb 's collar . , in their series of 12 pediatric patients with bulbar or posterior urethral stenosis , identified a sort of bimodal distribution of age at presentation , with 50% of patients presenting during the first year of life , whereas 5 of the remaining 6 patients presented after the age of 11 . this late presentation may represent a delayed presentation of a congenital stricture , or may be due to a forgotten urethral trauma or asymptomatic inflammation . clinical presentation may be variable and , often , may be related to the use of blind urethral catheterization in these patients . chronic urinary retention and bilateral hydronephrosis may be a presenting feature in some patients , as in our case . late presentation of cobb 's collar ( caused by a persistence of urogenital membrane ) can be differentiated from acquired stricture by the absence of the characteristic folds between copum anomalies and verumontanum . pediatric urologists continue to debate whether the cobb 's collar or moormann 's ring represents a true urethral stricture or not . some consider cobb 's collar to be a benign physiological narrowing of the bulbar urethra , easily passable with a cystoscope , although cobb describes a series of bulbar strictures in a pediatric population in his original paper . so , although rare , cobb 's collar has to be considered in the differential diagnosis of progressive urinary flow obstruction and enuresis , especially in pediatric and adolescent patients where acquired traumatic , iatrogenic , or inflammatory strictures are more common . radiologically indistinguishable dysfunctional voiding due to involuntary contraction of urethral sphincter or pelvic floor muscles and hinman syndrome ( functional non - neurogenic bladder ) are also important differential diagnoses to be considered . contrast - enhanced conventional radiological studies can identify this anomaly and also be helpful in postoperative follow - up of this entity which can be easily treatable by minimally invasive endoscopic corrective surgery . familial occurrence of cobb 's collar is exceedingly rare and only few cases are reported in the literature , indicating that this entity may have a familial basis . screening for family members is an important part of managing these patients . in the absence of traditional risk factors if the biopsy of the stricture and histological examination shows a normal urethral mucosa and the absence of any inflammation or fibrosis , then cobb 's collar becomes the likely diagnosis . from the limited data available and from the cases reported here , it is clear that urethral strictures often present with an unknown etiology . although extremely uncommon , in cases of adolescent or pediatric patients presenting with symptoms of progressive urinary obstruction and enuresis with or without urinary tract infection and/or hematuria , a bulbar urethral obstruction such as cobb 's collar should be considered .

most urethral strictures arise from iatrogenic , traumatic , or inflammatory causes . we report the familial occurrence of a congenital bulbar urethral stricture in two brothers . retrograde and voiding cystourethrography was performed . a cobb 's collar was diagnosed after radiological and endoscopic evaluation in both cases and was successfully managed with urethroplasty . cobb 's collar is a rarely recognized cause of a membranous stricture of bulbar urethra that can lead to several urinary problems . in cases of adolescent and young adults presenting with symptoms of progressive urinary obstruction and enuresis with or without urinary tract infection , cobb 's collar can be seen as a minor constriction in the bulbar urethra , but is not frequently symptomatic , and the familial occurrence of such a stricture is even rarer .

Introduction Case Reports Case 1 Case 2 Discussion Conclusion