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objectiveikzf1 encoding ikaros , an essential regulator of lymphopoiesis and immune homeostasis , has been implicated in the development of childhood acute lymphoblastic leukemia ( c - all ) . because recent genome - wide association ( gwa ) studies have linked a region of the 3-utr of ikzf1 with c - all susceptibility , we tested whether ikzf1 is associated with the autoimmune disease type 1 diabetes.research design and methodsrs10272724 ( t > c ) near ikzf1 at 7p12 was genotyped in 8,333 individuals with type 1 diabetes , 9,947 control subjects , and 3,997 families of european ancestry . association was tested using logistic regression in the case - control data and by the transmission disequilibrium test in the families . expression data for ikzf1 by rs10272724 genotype were obtained using quantitative pcr of mrna / cdna generated from peripheral blood mononuclear cells from 88 individuals , whereas expression data for five other neighboring genes were obtained from the online genevar dataset.resultsthe minor allele of rs10272724 ( c ) was found to be protective from type 1 diabetes ( odds ratio 0.87 [ 95% ci 0.830.91 ] ; p = 1.1 1011 ) . rs10272724 was not correlated with levels of two transcripts of ikzf1 in peripheral blood mononuclear cells.conclusionsthe major susceptibility genotype for c - all confers protection from type 1 diabetes . our finding strengthens the link between autoimmunity and lymphoid cancers . further investigation is warranted for the genetic effect marked by rs10272724 , its impact on ikzf1 , and the role of ikaros and other family members , ailios ( ikzf3 ) and eos ( ikzf4 ) , in autoimmunity .

a total of 8,333 type 1 diabetes cases , 9,947 controls , and 3,997 families were genotyped at rs10272724 using taqman ( supplementary methods ) . cases were diagnosed with type 1 diabetes before 17 years of age ( mean age at diagnosis = 7.8 years ) and were from the juvenile diabetes research foundation / wellcome trust diabetes and inflammation laboratory genetic resource investigating diabetes study ( http://www.childhood-diabetes.org.uk/grid.shtml ) . controls were obtained from the british 1958 birth cohort ( n = 6,899 ; http://www.cls.ioe.ac.uk/studies.asp?section=000100020003 ) and the wtccc uk blood service ( ukbs ) sample collection ( n = 3,048 ) ( 10,12 ) . stata version 10 ( statacorp lp , college station , tx ) was used to perform association analyses ( http://www.stata.com ) . rs10272724 was in hardy - weinberg equilibrium in unaffected parents and control subjects ( p > 0.05 ) . case - control data were modeled using logistic regression , with disease status as the outcome variable and counts of the minor allele ( coded 0 , 1 , and 2 ) as the independent variable , assuming a multiplicative allelic effects model . families were analyzed using the transmission disequilibrium test ( 13 ) ( supplementary methods ) . the interdependency of rs10272724 and rs4948088 in the 7p12 region was examined by stepwise logistic regression . peripheral blood mononuclear cells ( pbmcs ) were purified from heparinized blood diluted 1:1 in pbs ( without ca and mg , gibco , invitrogen , carlsbad , ca ) , and 15 ml aliquots were layered onto 10 ml aliquots of lympholyte ( cedarlane laboratories ltd . , burlington , ontario ) followed by centrifugation at 800 g for 20 min at room temperature . the harvested pbmc layer was washed twice with ice - cold pbs and centrifuged at 300 g for 10 min at 4c . pellets were resuspended in trizol ( invitrogen ) and stored at 80c in aliquots of 10 10 cells / ml . total rna from 1 10 pbmcs in trizol was prepared using chloroform extraction followed by purification with the rneasy mini kit ( qiagen , hilden , germany ) , according to the manufacturer s instructions . rna quality was assessed using an agilent 2100 bioanalyser , and concentration was evaluated by nanodrop ( thermo scientific , waltham , ma ) . first strand dna synthesis was carried out on 1 g of rna using superscript iii rt kit and oligo - dt ( invitrogen ) . quantitative ( q)pcr primers and probe were designed to two transcripts of ikzf1 , herein referred to as qpcr assays contained 5 l of 1:10 dilution of oligo - dt primed cdna , prepared as described above , in 20 l assays . cycle threshold ( ct ) values were measured using a 7900ht abi prism ( applied biosystems , carlsbad , ca ) and analyzed using sds v2.1 software ( abi ) . qpcr reactions were run in triplicate , and the ct for the transcript level qpcrs was calculated using the ikzf1 isoform 1 or isoform 2 qpcr ct value minus the single copy gene 2 microglobulin qpcr ct value . expression values were compared via one - way anova using prism software ( graphpad software inc . , la jolla , ca ) . correlation between rs10272724 and expression in three types of cell lines ( primary fibroblasts , epstein barr virus - immortalized lymphoblastoid cell lines , and phytohemagglutinin - stimulated primary t - cells ) derived from umbilical cord samples of 75 newborns of western european origin via the gencord project was examined in silico ( 14 ) using the publicly available hapmap online gene expression variation ( genevar ) resource ( http://www.sanger.ac.uk/resources/software/genevar/ ) . nine probes that passed quality control assessment ( supplementary table 1 ) were evaluated for correlation of mrna expression with rs10272724 genotype by linear regression using the genevar 2.0.1 java tool ( 15 ) ( supplementary methods ) . a p < 0.0056 significance threshold was used within each cell type based on a bonferroni correction for testing nine transcripts . blood for qpcr experiments was collected from 88 nondiabetic donors ( rs10272724 : 44 tt , 33 ct , 11 cc ) selected from a pregenotyped bioresource and processed within 4 hours ( www.cambroidgebioresource.org.uk ) . all dna samples were collected with ethical approval from the national health service cambridgeshire research ethics committee . written consent was obtained from all individuals . a total of 8,333 type 1 diabetes cases , 9,947 controls , and 3,997 families were genotyped at rs10272724 using taqman ( supplementary methods ) . cases were diagnosed with type 1 diabetes before 17 years of age ( mean age at diagnosis = 7.8 years ) and were from the juvenile diabetes research foundation / wellcome trust diabetes and inflammation laboratory genetic resource investigating diabetes study ( http://www.childhood-diabetes.org.uk/grid.shtml ) . controls were obtained from the british 1958 birth cohort ( n = 6,899 ; http://www.cls.ioe.ac.uk/studies.asp?section=000100020003 ) and the wtccc uk blood service ( ukbs ) sample collection ( n = 3,048 ) ( 10,12 ) . stata version 10 ( statacorp lp , college station , tx ) was used to perform association analyses ( http://www.stata.com ) . rs10272724 was in hardy - weinberg equilibrium in unaffected parents and control subjects ( p > 0.05 ) . case - control data were modeled using logistic regression , with disease status as the outcome variable and counts of the minor allele ( coded 0 , 1 , and 2 ) as the independent variable , assuming a multiplicative allelic effects model . families were analyzed using the transmission disequilibrium test ( 13 ) ( supplementary methods ) . the interdependency of rs10272724 and rs4948088 in the 7p12 region was examined by stepwise logistic regression . peripheral blood mononuclear cells ( pbmcs ) were purified from heparinized blood diluted 1:1 in pbs ( without ca and mg , gibco , invitrogen , carlsbad , ca ) , and 15 ml aliquots were layered onto 10 ml aliquots of lympholyte ( cedarlane laboratories ltd . , burlington , ontario ) followed by centrifugation at 800 g for 20 min at room temperature . the harvested pbmc layer was washed twice with ice - cold pbs and centrifuged at 300 g for 10 min at 4c . pellets were resuspended in trizol ( invitrogen ) and stored at 80c in aliquots of 10 10 cells / ml . total rna from 1 10 pbmcs in trizol was prepared using chloroform extraction followed by purification with the rneasy mini kit ( qiagen , hilden , germany ) , according to the manufacturer s instructions . rna quality was assessed using an agilent 2100 bioanalyser , and concentration was evaluated by nanodrop ( thermo scientific , waltham , ma ) . first strand dna synthesis was carried out on 1 g of rna using superscript iii rt kit and oligo - dt ( invitrogen ) . quantitative ( q)pcr primers and probe were designed to two transcripts of ikzf1 , herein referred to as qpcr assays contained 5 l of 1:10 dilution of oligo - dt primed cdna , prepared as described above , in 20 l assays . cycle threshold ( ct ) values were measured using a 7900ht abi prism ( applied biosystems , carlsbad , ca ) and analyzed using sds v2.1 software ( abi ) . qpcr reactions were run in triplicate , and the ct for the transcript level qpcrs was calculated using the ikzf1 isoform 1 or isoform 2 qpcr ct value minus the single copy gene 2 microglobulin qpcr ct value . expression values were compared via one - way anova using prism software ( graphpad software inc . , la jolla , ca ) . correlation between rs10272724 and expression in three types of cell lines ( primary fibroblasts , epstein barr virus - immortalized lymphoblastoid cell lines , and phytohemagglutinin - stimulated primary t - cells ) derived from umbilical cord samples of 75 newborns of western european origin via the gencord project was examined in silico ( 14 ) using the publicly available hapmap online gene expression variation ( genevar ) resource ( http://www.sanger.ac.uk/resources/software/genevar/ ) . nine probes that passed quality control assessment ( supplementary table 1 ) were evaluated for correlation of mrna expression with rs10272724 genotype by linear regression using the genevar 2.0.1 java tool ( 15 ) ( supplementary methods ) . a p < 0.0056 significance threshold was used within each cell type based on a bonferroni correction for testing nine transcripts . blood for qpcr experiments was collected from 88 nondiabetic donors ( rs10272724 : 44 tt , 33 ct , 11 cc ) selected from a pregenotyped bioresource and processed within 4 hours ( www.cambroidgebioresource.org.uk ) . all dna samples were collected with ethical approval from the national health service cambridgeshire research ethics committee . written consent was obtained from all individuals . the minor allele of rs10272724 ( t > c ) near ikzf1 was protective from type 1 diabetes ( odds ratio [ or ] 0.87 [ 95% ci 0.830.91 ] , p = 4.8 10 ; table 1 , supplementary table 2 ) . no evidence of association of rs10272724 with sex or age - at - diagnosis was obtained ( p > 0.1 ) . some samples overlapped with those used in the previous gwa study for type 1 diabetes ( 10 ) . however , in each of the sample sets used by barrett et al . ( 10 ) and in our unique samples , the effect is in the same direction and the overall significance is increased , suggesting the effect is not solely attributable to the original samples ( table 1 ) . the minor allele of rs10272724 was also protective in the families with type 1 diabetes , i.e. , under - transmitted to affected offspring ( table 1 ; relative risk 0.87 [ 95% ci 0.810.93 ] , p = 7.6 10 ) . combined , these results indicate that the minor allele , c , of rs10272724 at 7p12.2 is convincingly associated with a reduced risk of type 1 diabetes ( p = 1.1 10 ) . ( 10 ) reported a replicated association with rs4948088 ( c > a ) with type 1 diabetes in the 7p12.1 region , 554 kb downstream of ikzf1 . however , this snp , marking a confirmed type 1 diabetes locus ( http://www.t1dbase.org/page/regions ) , is not in ld with rs10272724 in ikzf1 ( d = 0.02 , r = 0.0001 ) , and we found by regression analysis that the two effects were independent as both snps improve a model with the other snp included ( p < 5.5 10 ) . association of rs10272724 ( t > c ) near ikzf1 in 8,333 type 1 diabetes cases , 9,947 control subjects , and 3,997 families with type 1 diabetes maf , minor allele frequency ; rr , relative risk . * no evidence of deviation from a multiplicative allelic effects model was obtained ( p = 0.33 ) , so p values assuming multiplicative allelic effects are reported . the overall p value was obtained by combining the p value from the case - control sets and the family transmission disequilibrium test using fisher s method for combined probability . ( 10 ) analyzed three datasets in their gwa study , two of which overlap with the samples in the current study . used the illumina 550 k snp chip to genotype rs10272724 in set 1 ( 4 ) . genotypes were 99.6% concordant between the illumina platform and taqman in the 3,850 cases and 3,772 controls genotyped using both technologies . affymetrix 500 k mapping array genotypes for neighboring snps were used to impute rs10272724 genotypes in set 2 by barrett et al . because rs10272724 was not genotyped by affymetrix . t1dgc , type 1 diabetes genetics consortium ; wtccc , wellcome trust case - control consortium . the relative abundance of two different sets of ikzf1 transcripts in mrna extracted from pbmcs isolated from 88 nondiabetic individuals was assessed using qpcr to determine the association of rs10272724 alleles with gene expression . no evidence of allele - specific expression was obtained ( p > 0.1 ; fig . the probe originally used ( illumina i d ilmn_1676575 ) contains two high - frequency snps in its target , rs62447207 ( g > t ) and rs62447208 ( c > g ) in ld with rs10272724 ( r = 0.76 ) , rendering it unreliable , as evidenced by the absent ikzf1 probe in the latest genevar data from the study by dimas et al . next , correlation of rs10272724 genotype and expression of nine transcripts from other genes within 1 mb of rs10272724 in three types of cell lines was tested using the latest genevar dataset by dimas et al . no correlation was observed between rs10272724 and expression of any of the five neighboring genes ( , supplementary fig . 1 ) . association of ikzf1 expression with rs10272724 ( t > c ) genotype in 88 unaffected individuals . ct were calculated using the ikzf1 isoform 1 or isoform 2 qpcr ct value minus the single copy gene 2 microglobulin ( b2 m ) qpcr ct value . we provide evidence that the minor allele of rs10272724 ( c ) , in near - perfect ld with the minor , c - all susceptible , allele of rs4132601 ( g ) , is protective from type 1 diabetes . of the nearby genes in the region , ikzf1 appeared to be the most likely candidate causal gene for type 1 diabetes in this region given its causal role in c - all and its known role in lymphocyte development . however , our analysis revealed that the previously reported correlation of ikzf1 mrna expression with rs4132601 may be inaccurate given complications with the probe used in the data accessed by papaemmanuil et al . furthermore , an ikzf1 genotype - phenotype correlation at rs10272724 was not confirmed by our qpcr analysis of pbmcs . ikzf1 is now an attractive candidate causal gene in type 1 diabetes , and a functional link between rs10272724 and ikzf1 may yet be elucidated on examining specific isoforms of ikaros . we note that our qpcr assay evaluated the expression of two transcripts conserved across 10 splice variants of ikzf1 , spanning six of the seven identified active and dominant - negative isoforms of the protein . in addition , many allele - specific effects on gene expression are known to be tissue or cell - type specific and may even be restricted to particular phases of development . another all - related gene , af4/fmr2 family member 3 ( aff3 ) , has been associated with type 1 diabetes ( 10 ) ( j. cooper , c.w . , and j.a.t . , unpublished observations ) , so our report also confirms a second genetic link between these diseases . aff3 has been implicated as a susceptibility allele in rheumatoid arthritis ( rs10865035 and rs9653422 ) ( 16 ) , further highlighting the connection between autoimmunity and lymphoid cancers . our finding with ikzf1 marks the third ikaros family member to be associated with type 1 diabetes , the others being the transcription factors ailios ( ikzf3 ) on chromosome 17q21.2 and eos ( ikzf4 ) on chromosome 12q13.2 ( 10,17 ) , whose targets include bcl-2 and foxp3 , respectively . ikaros family members interact to coordinate functions of immunologic development and homeostasis ( 18 ) , so it will be important to explore the effect of these interactions in disease etiology . the more than 30 reported interaction partners of the ikaros family members ( http://www.t1dbase.org ) suggest the role of ikzf1 in type 1 diabetes could be subtle and yet far reaching . interactions with the histone deacetylase and chromodomain - helicase - dna - binding families of proteins suggest chromosome remodeling events could be involved ( 11,19 ) . ikaros interactions with the notch ( 18 ) and stat ( 20 ) protein families also suggest that signaling events that affect t - cell activation and maturation could affect the ultimate development of a diabetogenic , leukemia - protected or nondiabetogenic , leukemia - susceptible t - cell repertoire . thus , further investigation is warranted to elucidate the phenotypic effect of the genetic feature marked by rs10272724 , its impact on ikzf1 , and the role of ikaros in autoimmunity .

aim . to revisit the role of first trimester homocysteine levels with the maternal and fetal outcome . methods . this was a cohort study comprising 100 antenatal women between 8 and 12 weeks of gestation . serum homocysteine levels were checked after overnight fasting . results . there were significantly elevated homocysteine levels among women with prior history of hypertensive disorders of pregnancy and prior second or third trimester pregnancy losses . there was no significant difference in homocysteine levels among women with previous gestational diabetes mellitus , preterm deliveries , or fetal malformations . homocysteine levels were significantly elevated in those who developed hypertensive disorder of pregnancy , oligohydramnios , and meconium stained amniotic fluid , had a pregnancy loss , or delivered a low birth weight baby . there was no significant difference in homocysteine levels for those who developed gestational diabetes mellitus . conclusions . increased first trimester serum homocysteine is associated with history of pregnancy losses , hypertensive disorders of pregnancy , and preterm birth . this is also associated with hypertensive disorders of pregnancy , pregnancy loss , oligohydramnios , meconium stained amniotic fluid , and low birth weight in the current pregnancy . this trial is registered with clinicaltrials.gov ctri/2013/02/003441 .

homocysteine is an amino acid which has sprung into prominence in the past few decades . elevated homocysteine has also served as an early marker for insulin resistance due to the effects of insulin on homocysteine metabolism and renal clearance . these relationships of homocysteine to disease states in the nonpregnant adult population have been extrapolated to link it to the pregnancy specific conditions of gestational diabetes mellitus and hypertensive disorders of pregnancy . homocysteine levels decline during pregnancy , and the levels are the lowest during second trimester of pregnancy and increase in the second half of the third trimester of pregnancy . hence , samples taken within strict time frame , such as 4 weeks ( between 8 to 12 weeks of gestation ) , would have a better success at correlating the homocysteine levels with the pregnancy outcome , by minimising the gestational age bound variation of homocysteine . in addition , most maternal complications such as hypertensive disorders of pregnancy and gestational diabetes mellitus develop much later , so timing the sample collection before 12 weeks would sufficiently predate the onset of complications . serum homocysteine levels in pregnancy have been linked to preeclampsia [ 46 ] , recurrent abortions [ 79 ] , and low birth weight . but evidence on this is conflicting with some studies stating that serum homocysteine values have no correlation to maternal and fetal outcome . hence , there is a need for this study to shed light on these conflicting opinions regarding the effect of homocysteine levels on both maternal and fetal outcomes . to correlate the levels of homocysteine in late first trimester of pregnancy ( 812 weeks ) with the maternal and fetal outcome of pregnancy , especially with regard to development of hypertensive disorders of pregnancy and gestational diabetes mellitus . to estimate the homocysteine levels in late 1st trimester ( 812 weeks ) and to correlate it with prior history of ( i ) first , second , and third trimester pregnancy losses , ( ii ) hypertensive disorders of pregnancy and gestational diabetes mellitus , ( iii ) fetal malformations , and ( iv ) preterm delivery and birth weight . to correlate the homocysteine levels with the present pregnancy parameters , namely , ( i ) hypertensive disorder of pregnancy , ( ii ) gestational diabetes mellitus , ( iii ) pregnancy loss , ( iv ) amniotic fluid volume and meconium staining of amniotic fluid , and ( v ) birth weight . it was a prospective observational cohort study comprising antenatal women who attended the antenatal outpatient department of a tertiary care university level health centre . the study was started after obtaining the institutional ethics committee approval and was conducted from august 2009 to july 2011 . 110 women were invited to participate in the study and 100 agreed to participate in the study . ten women refused to participate in the study as they were from distant places from our institute and hence could not come in the early morning in fasting state for the homocysteine test . subjects were enrolled at 8 to 12 weeks gestation and known diabetics or hypertensives on treatment were excluded . at enrollment , a questionnaire was collected regarding age , period of gestation , parity , abortions with respective period of gestation and whether induced or spontaneous , history of previous fetal malformations , history of hypertensive disorders of pregnancy or gestational diabetes mellitus in previous pregnancies , and birth weight of previous children and whether the previous deliveries were term or preterm births . venous blood samples were taken after overnight fasting for serum homocysteine estimation and serum separated by centrifugation and sera assayed for homocysteine by chemiluminescence assay technique using a well - calibrated fully automated chemiluminescence assay system the advia centaur cptm immunoassay system ( siemens , germany ) which is an automated , random access direct chemiluminescent immunoassay analyzer . routine examination included blood pressure recordings , pedal edema , urine albumin and sugar , symphysio fundal height , and clinically amniotic fluid level status . glucose challenge test was done at 32 weeks of pregnancy , and if abnormal , 100 gram oral glucose tolerance test ( ogtt ) was done to diagnose gestational diabetes mellitus . antenatal ultrasound was done if there was clinical suspicion of iugr ( e.g. , symphysio fundal height less than 4 cm than corresponding to period of gestation ) , and in case of malpresentations , hypertensive disorders of pregnancy ( and other risk factors for iugr ) and before induction of labour . doppler ultrasound of umbilical and middle cerebral artery was done in case of iugr and preeclampsia to do assess the risk of fetal morbidity in these cases . statistical analysis was done using graphpad instat software ; using unpaired t - test for comparison of two parametrically distributed groups of data , unpaired t - test with welch correction was used for comparison of two parametrically distributed groups of data with unequal variances . mann - whitney u test was used for comparison of two nonparametrically distributed groups of data , and one way anova was used for more than two parametrically distributed groups of data . kruskal - wallis test was used for comparison of more than two parametrically distributed groups of data . p value of 0.05 was taken as significant and 0.001 was taken as highly significant . of the 100 antenatal women recruited , 10 were lost to follow up thus leaving 90 subjects whose outcomes were analysed . the ages of the subjects were in the range of 21 to 33 years with a mean age of 24.76 2.6 years . the bmi of the subjects ranged from 15.5 kg / m to 33.2 kg / m and the mean was 21.70 6.23 there was no correlation between the homocysteine levels and the age , bmi , the period of gestation , or the number of folic acid tablets taken by the subjects . of the recruited 100 women , 14 women had past history of hypertensive disorders of pregnancy , 5 women had previous gestational diabetes mellitus , 6 had a previous preterm delivery , and 2 had a previous fetal malformation . there was significantly elevated homocysteine levels ( p = 0.0359 ) among women with prior history of hypertensive disorders of pregnancy . however , the difference in homocysteine levels between those with and without past history of gestational diabetes mellitus was not statistically different ( p = 0.054 ) . there was no statistically significant difference of homocysteine levels with history of prior preterm deliveries ( p = 0.8348 ) or with history of previous fetal malformation ( p = 0.078 ) ( table 1 ) . of the 100 antenatal women , 27 women had previous one and 9 women had previous two first trimester pregnancy losses , 9 women had previous one and 3 had previous two second trimester pregnancy losses , and 9 women had previous one third trimester pregnancy loss . there was no statistically significant elevation of homocysteine levels in those with history of prior first trimester pregnancy losses ( p = 0.1687 ) . there was statistically significant elevation of homocysteine levels in those women with history of prior second trimester pregnancy losses ( p = 0.0307 ) . there was highly significant elevation of homocysteine levels in those women with history of prior third trimester pregnancy losses ( p < 0.0001 ) . comparing women with no history of prior pregnancy losses in any trimester and those with pregnancy losses in any trimester , there was highly significant elevation of homocysteine levels in the latter group ( table 2 ) . there was no significant correlation between prior birth weights and the serum homocysteine levels ( table 2 ) . the present pregnancy outcome of the 90 women who completed follow - up ( 10 women were lost to follow up ) was then analysed . of the 90 women , 18 developed hypertensive disorders of pregnancy ( hdp ) and 7 developed gestational diabetes mellitus ( gdm ) ; 2 patients had a fetus with a congenital malformation and 8 patients had a pregnancy loss ( including 2 stillbirths ) . of the 84 antenatal women whose delivery outcomes were analysed : 18 women had oligohydramnios , 17 women had meconium stained amniotic fluid , and 11 women delivered low birth weight babies ( birth weight < 2500 gm ) . the homocysteine levels were statistically elevated in those who went on to develop hdp than those who did not develop hdp ( p = 0.0011 ) . there was no significant difference ( p = 0.6312 ) in homocysteine levels between those who developed and those who did not develop gdm . the power of the study to detect a difference in gdm was 0.42 ( table 3 ) . there was significant elevation in homocysteine levels ( p = 0.0002 ) among those who suffered a pregnancy loss when compared to those who had a live birth in the current pregnancy . there was no statistically significant difference in homocysteine levels ( p = 0.6621 ) between those who had a fetus with a congenital malformation and those who had a morphologically normal fetus . there was statistically significant increase in homocysteine levels in women with oligohydramnios on comparing with women with normal amniotic fluid levels ( p < 0.001 ) , and women with meconium stained amniotic fluid had higher homocysteine levels than those with clear amniotic fluid ( p = 0.0037 ) . the serum homocysteine levels were significantly elevated ( p = 0.0224 ) for those women who gave birth to low birth weight babies than those whose babies weighed above 2500 grams at birth ( table 4 ) . serum homocysteine is not significantly correlated with the age of the subjects , which is in agreement with another indian study by das et al . . serum homocysteine levels are not significantly associated with body mass index , in concordance with a study by han et al . . there was no significant variation with the mean period of gestation at which the samples were taken with the serum homocysteine concentrations . this may be due to the factor that the time window in which the samples were taken itself was short , and the study by murphy et al . and dodds et al . on the homocysteine levels longitudinally through pregnancy suggests that the homocysteine levels may be more stabilised from 8 weeks of gestation onward up to the middle of second trimester ; there is a reduction in homocysteine levels prior to 8 weeks and a rise in homocysteine levels in the third trimester . there was no significant correlation between homocysteine levels and the number of folic acid tablets taken in correlation with the prior studies [ 4 , 16 ] . the difference in homocysteine levels in those with prior first trimester losses did not reach statistical significance in this study . however , when all prior pregnancy losses were taken together , the difference in homocysteine levels was statistically significant ; the same statistical significance held true with those who had prior second or third trimester losses . this can be compared to other studies , which have also showed that prior early pregnancy losses [ 8 , 9 , 17 ] and stillbirths are reflected in higher homocysteine levels . those with prior hypertensive disorders of pregnancy also had increase in homocysteine levels , in concordance with other studies [ 18 , 19 ] . prior preterm birth was not significantly associated with homocysteine levels , in contrast to a study by kramer et al . which saw an association of preterm birth with higher homocysteine levels , but the proposed mechanism of decidual vasculopathy of the placenta due to higher homocysteine level could not be proven by that study . the serum homocysteine levels was not different between those who had a previous fetal malformation in contrast with the hordaland homocysteine study which showed an increased incidence of malformations , particularly neural tube defects , in those mothers with elevated homocysteine levels . there was a declining trend of prior birth weights with higher homocysteine levels confirmed by other studies . with reference to the index pregnancy , the serum homocysteine levels were significantly different between those who went on to develop hypertension during their pregnancy , and those who remained normotensive , in conformance with prior studies [ 57 , 20 ] . post priori power calculation has shown that the power to detect a difference in hypertensive disorder of pregnancy was 0.95 which indicates that the present study was adequately powered to show that elevated homocysteine is associated with development of hypertensive disorders of pregnancy . the serum homocysteine levels were not significantly different between those who developed gestational diabetes mellitus and those who maintained normal glucose tolerance , similar to a study by idzior - walu et al . however , it is in contrast to a study by tarim et al . who found a significantly elevated homocysteine level in antenatal women with gestational diabetes mellitus . nevertheless , much conclusion can not be drawn from the present study as the post priori power calculated was 0.42 only . the serum homocysteine levels were not different between those who had fetuses with congenital malformations and normal fetuses , in conformance with the study of wang et al . this is in concordance with several other studies which also showed an increase in the rate of spontaneous abortions and stillbirths [ 18 , 24 ] in pregnancies with elevated homocysteine levels . the difference in homocysteine levels between those with oligohydramnios and normal amniotic fluid level status and those with clear and meconium stained amniotic fluid has been shown to be statistically significant in this study . however , there is a lack of previous studies to show this association . however , this can be taken as an extrapolation of the higher incidence of preeclampsia [ 57 , 20 , 25 ] , fetal growth restriction , and impairment of placental transport that has been documented in previous studies . this can also be taken as an extrapolation of the increased incidence of hypertensive disorders of pregnancy and its associated placental insufficiency . the difference in serum homocysteine levels between those with low birth weight babies and those whose birth weight was above 2500 grams was statistically significant . this is in conformance with most studies [ 26 , 28 ] but one study contradicted stating that there was no statistical difference . the a priori power calculation in the study was based on prior study on serum homocysteine levels in hypertensive disorders of pregnancy . due to paucity of data on serum homocysteine levels in gestational diabetes the post priori power calculation was 0.42 which indicates that this study may not have had the power to detect a difference in homocysteine levels in gestational diabetes . the number of cases was small and definite conclusions can not be derived from this study . however , this study is the first of its kind in this ethnic population in a developing country . further studies will be needed to strengthen these conclusions . though the number of folic acid tablets taken prior to the sample collection for homocysteine is quantified ( by memory recall of the patients ) , the number of folic acid tablets taken by the patients after the sample collection during the rest of the pregnancy may be a confounding factor , especially due to the fact that though folic acid is routinely recommended during first trimester , the focus shifts to iron during the second and third trimester , notwithstanding the fact that many iron preparations contain folic acid in addition to iron . this is a limitation of this study since homocysteine values vary with folate and b12 levels . serum homocysteine levels in late first trimester ( 8 to 12 weeks ) of pregnancy are significantly associated with prior pregnancy losses , particularly in the second and third trimesters and with prior hypertensive disorders of pregnancy . with reference to the current pregnancy , increased serum homocysteine levels are also significantly associated with hypertensive disorders of pregnancy , pregnancy loss , oligohydramnios , meconium stained amniotic fluid , and low birth weight . however , raised homocysteine levels are not significantly associated with body mass index or with gestational diabetes mellitus and fetal malformations , neither in the past pregnancies nor in the current pregnancy .

isolated bilateral deafness is a rare but possible symptom of vertebrobasilar ischemia , primarily due to occlusion of the anterior inferior cerebellar arteries or their branch , the internal auditory artery . we reported on uncommon case of sudden bilateral sensorineural hearing loss without typical neurological symptoms resulting from vertebrobasilar ischemia . we performed the available examinations , including otoscopy , laboratory tests , and pure tone audiogram , however we were not able to identify the cause of bilateral sensorineural hearing loss . brain magnetic resonance image showed the cerebellar infarction of the posterior inferior cerebellar artery territory . brain magnetic resonance angiography showed bilateral vertebral and basilar artery occlusion . we suggest vertebrobasilar ischemia as a cause of sudden isolated deafness .

most sudden onset sensorineural hearing loss occurs unilaterally , with an the incidence of bilateral involvement of less than 5%.1 ) various causes , such as infection , vascular event , coagulation disorders , neoplasm , and demyelinating disease , are related to sudden sensorineural hearing loss , however in most cases the causes are idiopathic.2 ) although rarely reported , deafness can also be attributed to vertebrobasilar ischemia.3 ) the internal auditory artery ( iaa ) provides the main blood supply to the cochlear nerve and cochlea . the iaa is usually a branch of the anterior inferior cerebellar arteries ( aica ) , however it could originate from the posterior inferior cerebellar arteries ( pica ) or directly from the basilar artery . due to its absolute absence of collateral blood supply and very high - energy metabolism , the inner ear is particularly vulnerable to vertebrobasilar ischemia.4 ) although most sudden hearing loss due to vertebrobasilar ischemia is associated with other combined neurologic signs or symptoms , vertebrobasilar ischemia can also develop as isolated sudden sensorineural hearing loss . we report on an uncommon case of sudden bilateral sensorineural hearing loss , related to vertebrobasilar occlusion without other neurologic deficit . a 64-year - old patient was admitted to the emergency room with the complaint of sudden bilateral hearing loss . two days earlier , the patient had suddenly developed vertigo and vomiting , and sequentially noticed bilateral hearing loss the following day . the patient had non - insulin dependent diabetes mellitus , hypertension , and a history of heavy smoking , but had no previous history of hearing impairment , head trauma , meningitis , autoimmune diseases , or ototoxic drugs . on neurological examination , no abnormal findings in the external auditory canal and eardrum were observed during examination using an otoscope . blood tests , including complete blood count , serum electrolyte , liver function test , urea nitrogen , creatinine , and high - sensitivity c - reactive protein ( hscrp ) showed normal results . pure tone audiogram showed severe sensorineural hearing loss of 73 decibels ( db ) on the left side and 86 db on the right side ( fig . brain magnetic resonance image diffusion weighted imaging ( mri dwi ) and mr angiography ( mra ) ( fig . 2 ) showed a multifocal cerebellar infarction in the bilateral pica territory and bilateral vertebral and basilar artery occlusion , respectively . occlusion of the right proximal vertebral artery and basilar artery , and severe stenosis of the left distal vertebral artery were observed on transfemoral cerebral angiography ( fig . we concluded that the bilateral iaa might be compromised with basilar artery occlusion , resulting in bilateral sensorineural hearing loss . sometimes , the iaa is also a branch of pica or basilar artery , whose occlusion would directly cause hypoperfusion of the iaa . the patient was treated with anticoagulation , heparin and warfarin for three months , and was then changed to aspirin . two weeks later , a follow up pure tone audiogram recorded improvement on both sides , to 58 db on the left and 71 db on the right ( fig . rapid notice of such an event and initiation of proper management are critical to prevention of grave results . although various neurological deficits , such as dysarthria , numbness , weakness , and ataxia , occur mainly in vertebrobasilar ischemia , isolated bilateral sensorineural hearing is possible , but rare.5 ) previous studies have reported incidence of vertebrobasilar occlusion in sudden sensorineural hearing loss as approximately 1.2% and only 1.4% of patients with vertebrobasilar occlusive disease had bilateral hearing loss.6 - 7 ) in this case , the cause of isolated bilateral sensorineural hearing impairment might be iaa territory ischemia . tranfemoral cerebral angiography also showed occlusion of the right proximal vertebral artery and basilar artery and severe stenosis of the left distal vertebral artery . in the current report , the mechanism of sudden bilateral hearing loss is that an atheromatous plaque within the basilar artery extended into the aica orifice and a profound degree of hypoperfusion in both aicas may have caused selective injury of the inner ear . another possible mechanism is that isolated bilateral sensorineural hearing impairment could be a symptom of basilar artery or pica occlusion . although the auditory system including inner ear and auditory nerve is usually supplied by the iaa , a branch of aica , the iaa can arise from the pica or directly from the basilar artery.3 ) the inner ear is vulnerable to ischemia for the following reasons . first , the inner ear is supplied from an end artery , while other auditory system , such as the auditory nerves in the internal auditory , have several anastomosing vessels . in addition , the cochlea requires higher energy for work than the vestibular structure.6 ) verebrobasilar ischemia could have a poor prognosis with severe truncal ataxia or evolve into locked - in syndrome or coma ; some patients have eventually died.8 - 9 ) there was no recommended treatment with anticoagulation in total stenosis of intracranial arteries , however , we began anticoagulation therapy for prevention of ongoing cerebral infarction and the patient had resolved by 2 weeks.10 ) as mentioned earlier , despite its rareness , occlusion or stenosis of the vertebrobasilar artery , aica , and pica could lead to bilateral sensorineural hearing loss . when attempting to determine the etiology of isolated bilateral deafness , it should be kept in mind that clinicians should consider the possibility of vertebrobasilar occlusive disorder especially in patients with risk factors for stroke and presenting with other neurologic signs .

micronutrients play an important role in the development of effective immune responses . this study characterised a populations exposed to schistosome infections in terms of the relationship between micronutrients and immune responses . levels of retinol binding protein ( rbp ; vitamin a marker ) , vitamin d , ferritin and soluble transferrin receptor ( stfr ) , and c reactive protein ( crp ) were related to levels of schistosome specific cytokines ( ifn- , il-4/5/10 ) in 40 zimbabweans ( 754 years ) exposed to schistosoma haematobium infection . 67.2% of the participants were deficient in vitamin d. rbp levels were within normal ranges but declined with age . the two indicators of iron levels suggested that although levels of stored iron were within normal levels ( normal ferritin levels ) , levels of functional iron ( stfr levels ) were reduced in 28.6% of the population . schistosome infection alone was not associated with levels of any of the micronutrients , but altered the relationship between parasite - specific il-4 and il-5 and levels of ferritin and stfr .

micronutrients are known to play an important role in health and the development of an effective immune system . in tropical and subtropical regions schistosomiasis is a global health burden with over 200 million people infected by one of five schistosoma trematode species [ 1 , 4 , 5 ] . schistosoma haematobium is the causative agent of urogenital schistosomiasis and is widely distributed in africa . simultaneously , according to the global progress report on vitamin and mineral deficiency , more than half of africa 's population lack critical vitamins and minerals . deficiencies in iron and vitamin a each rank among the top 10 leading causes of death in developing countries through disease . a recent study in nigeria showed that infection with s. haematobium affected growth and nutritional status of children . it is clear that micronutrient supplementation though programmes such as expanded programme of immunisation ( epi ) and child health days can help reduce under 5 mortality , which is the stated aim of millennium development goal 5 . with growing calls for integrated approaches to improving human health , it is important to characterise the interaction between micronutrient deficiencies and the immune response to schistosomiasis so that public health programs can plan their interventions accordingly . helminth infection including infection by schistosomes , modulates the host immune response , manifesting as diminished allergic responses , amelioration of autoimmune disease , and chronic parasitic infection [ 911 ] . immunomodulation is mediated by regulatory t cells ( treg ) through direct contact stimulation and il-10 production [ 12 , 13 ] . while the switch to th2 which occurs during helminth infection is an effective antiparasitic response , it is unclear whether superimposition of regulatory responses primarily benefits the worms or the host . downregulation of the inflammatory response would reduce host mediated immunopathology but also reduce protection [ 9 , 14 ] . these effects are seen as a diminished allergic response , amelioration of autoimmune disease and chronic parasitic infection . traditionally vitamin a has been known for its role in vision , with deficiency resulting in xerophthalmia , which is the leading cause of preventable childhood blindness . however , it has a wide range of physiological functions and is essential for haematopoiesis and prevention of anaemia , as well as immune function . it is acquired from foods such as liver , milk , cheese , eggs , green leaves , carrots and ripe mangos . vitamin a has now been implicated in the development of th2 , th17 and treg responses through the activation of retinoid receptors . retinoic acid activates the foxp3 transcription factor , which stimulates the development of nave t cells into treg[1517 ] . hypovitaminosis a is an immunodeficient state linked to decreased antibody production , typically diminished th2 antibodies ige , igg1 , and iga . it is produced in the skin when 7-dehydrocholesterol reacts with uvb radiation to form vitamin d3 , which modified in the liver to form 25(oh ) vitamin d3 , and converted to its active metabolite 1,25(oh)2 vitamin d3 in the kidney . they are then metabolised by the liver in the same manner as cutaneously derived vitamin d3 . a role has been suggested for vitamin d in diseases with an immunological aetiology such as psoriasis , multiple sclerosis and diabetes mellitus . it suppresses the th1 cytokines ifn- and il-2 , and upregulates il-4 to create a th2 polarisation . vitamin d can stimulate treg through production of tgf-1 and cd25 expression by cd4 t cells [ 2224 ] . it also diminishes expression of dendritic cell ( dc ) costimulatory markers cd40 , cd80 , and cd86 , again linked to treg induction [ 14 , 23 ] . anaemia affects 1.62 billion people worldwide , and around 500 million of those people have iron deficiency anaemia . a causal relationship between infection with s. japonicum and iron deficiency anaemia has been established . it is linked to increased infectious mortality and morbidity , and can itself be caused by chronic infection [ 27 , 28 ] . its relationship with infection is complex as both pathogen and host use body iron stores . it has been shown that iron supplementation during active infection can increase the infectious load of some pathogens [ 27 , 29 ] . experimental studies on mice have found that those with high iron indices had a significantly increased fibrosis around egg granulomata . iron deficiency is associated with igg1 , ige , and treg responses whereas iron supplementation has been linked to th1 responses and decreased il-10 [ 30 , 31 ] . the soluble transferrin receptor ( stfr ) is a diagnostic tool for differentiating between iron deficiency anemia ( ida ) and anemia of chronic disease since ferritin levels reflect amounts of stored iron while the stfr reflects the functional iron compartment . a few studies have shown a recent review of data collected in zimbabwe between 1980 and 2006 showed that a significant proportion of preschool children , school children , and adult women ( lactating or pregnant ) experienced malnutrition with significant proportions of these groups suffering from vitamin a and iron deficiencies . the aim of this study was to determine the relationship between the micronutrients vitamin a , d , and iron as well as a measure of inflammatory responses c - reactive protein ( crp ) and schistosome - specific cytokine levels in zimbabweans exposed to s. haematobium infection . the study received ethical and institutional approval from the medical research council of zimbabwe and the university of zimbabwe , respectively . permission to conduct the work in this province was obtained from the provincial medical director . informed consent / assent was obtained from all participants or their parents / guardians prior to enrolment into the study . project aims and procedures were explained to the community , school children , and their teachers prior the study , and survey was conducted amongst all compliant participants . after sample collection , all participants were offered treatment with the standard dose of 40 mg / kg body weight of the antihelminthic drug praziquantel . the study was conducted in two rural villages in the mashonaland east province of zimbabwe ( 3130e ; 1745s ) where s. haematobium is endemic . participants were part of a larger immunoepidemiology study which was carried out between 2002 and 2005 , and the study area is described in detail elsewhere . drinking water is collected from open wells while bathing and washing is conducted in two main rivers in the villages . most families maintain a garden located near the river where water is collected for watering the crops and the schools surveyed were all in close proximity to rivers . all samples used in this study were obtained at baseline in 2002 were selected using following criteria : ( 1 ) participants should be life - long residents in this area ( assessed by questionnaire ) , ( 2 ) should not have received antihelminthic treatment prior this study , ( 3 ) should have provided at least two urine and 2 stool samples on consecutive days to allow parasitological diagnosis , ( 4 ) should have been test negative for soil transmitted helminth and s. mansoni as well as negative for hiv and plasmodium falciparum , ( 5 ) should have provided a blood sample to obtain sera . furthermore , only sera samples were used for these analyses , which have not been used previously and therefore were defrosted for the first time . following these criteria samples from 40 people aged 754 years ( 13 male , 27 female ) data were subsequently separated into 3 age groups : 710 years ( n = 6 ) , 1120 years ( n = 23 ) , 21 + years ( n = 11 ) , which represent a typical age - infection profile for s. haematobium as shown in figure 1(a ) . parasitology samples ( at least 2 urine and 2 stool samples collected on 3 three consecutive days ) and 20 ml of venous blood were collected from each participant . stool samples were processed following the kato - katz procedure to detect s. mansoni eggs and other intestinal helminths , while the urine filtration method was used to detect s. haematobium eggs in urine samples . serum samples obtained from 20 ml of venous blood from each participant were frozen and stored in duplicate at 20c in the field and transferred to a 80c freezer in the laboratory . one complete set of the samples was subsequently transported frozen from zimbabwe to the uk , stored at 80c and defrosted for the first time for use in this study . small aliquots of blood were used to prepare thick and thin smears for the microscopic detection of plasmodium parasites . the parasite - specific cytokines ifn- , ( marker for th1 responses ) il-4 , il-5 ( markers of th2 responses ) , and il-10 ( marker for regulatory responses ) were measured by enzyme linked immunosorbent assays ( elisa ) in supernatants obtained after stimulation of whole blood samples using cercarial , egg , and adult schistosome antigens following published methods . spontaneous cytokine production was determined in unstimulated controls containing media alone while the mitogen concanavalin a ( cona ) was used as a positive control for the restimulations . values of cytokines obtained from the media alone incubations were subtracted from those of the antigen - specific restimulations to remove the effects of background cytokine production in the statistical analyses . micronutrients and c reactive protein ( crp ) were measured using enzyme linked immunosorbent assay ( elisa ) kits according to manufacturers ' instructions . serum transferrin receptor ( stfr ) is a marker of iron deficiency and is required for lymphocyte activation and proliferation . ferritin is a marker of iron status , but rises with inflammation [ 27 , 38 ] and this was measured by an elisa kit from bioquant ( cat . crp is an inflammatory marker and was measured by an elisa kit from anogen ( cat . retinol binding protein a measure of vitamin a status was assayed using an elisa kit from phoenix pharmaceuticals ( cat . # ek-028 - 28 ) , and 25(oh ) vitamin d was used to assess the inactive vitamin d status although through a kit from immunodiagnostik ( cat . vitamin d status was described using previously published ranges ( replete 50.00 nmol / l , mild deficiency 25.0049.99 nmol / l , moderate deficiency 12.5024.99 nmol / l , severe deficiency 12.49 nmol / l ) . the world health organisation reference range for ferritin was used ( female normal range 15.0150.0 g r&d systems provided a 2.597.5 percentile range ( 8.728.1 nmol / l ) for stfr from a survey of 225 ethnically diverse participants of both sexes . their mean value for afro - carribeans was significantly higher than for other ethnic groups . there is no published reference range for rbp , although the world health organisation has produced retinol reference ranges for use in public health [ 43 , 44 ] . the ratio of stfr / log ferritin ( stfr - f index ) has been suggested as an alternative estimate of body iron , so this was also calculated in this study and used in the statistical analyses . for the statistical analyses , host infection intensity was recorded into infection status , that is , infected and uninfected , cytokine absorbencies were square root transformed , and levels of all micronutrients were log transformed to satisfy the assumptions of parametric tests . in order to determine if the relationship between micronutrients and immune responses differed between schistosome infected versus uninfected people , a multivariate analysis of variance ( manova ) was conducted . the dependent variables were the transformed micronutrient data and the independent variables were cytokine levels , infection status ( infected / uninfected ) age ( categorical ( 710 years , 1120 years , 21 + years ) ) , sex ( categorical male / female ) . the effects of interactions between infection status and micronutrients were also included in the manova model . sequential sums of squares were used to calculate the test statistics so that the potentially confounding effects of all other variables could be allowed for testing for the effects of infection status which was entered last in the single effects list . schistosome infection prevalence in the study population was 60% ( 95% ci : 4375% ) and the mean infection intensity was 39.3 eggs/10 ml urine ( sem = 13.5 ) with a range of 0362 eggs/10 ml urine . infection intensity followed the typical schistosome age - infection pattern , rising with age to a peak in childhood and declining thereafter ( figure 1(a ) ) . the study population had a mean rbp of 0.23 ng / ml with a range of 00.63 ng / ml . 25(oh ) vitamin d titres in this population were low when compared to published values with 32.8% ( n = 12 ) of the population being classified as vitamin d replete ( 50.00 nmol / l ) ; 17.9% ( n = 7 ) were mildly deficient ( 2549.90 nmol / l ) , 10.3% ( n = 4 ) were moderately deficient ( 12.5024.90 nmol / l ) , and 38.5% ( n = 15 ) were severely deficient ( 12.49 levels of crp were within the normal range while 28.6% ( n = 10 ) of the participants had elevated stfr based on the 95 percentile data provided with the assay as detailed in the methods section . the statistical analyses showed that sex affected only levels of ferritin , which was significantly lower in females and did not have a significant effect on levels of any of the other micronutrients ( table 1 ) . age significantly affected levels of rbp , with rbp levels falling with age ( r = 0.315 , p = 0.033 ) as shown in figure 1 , but did not affect levels of any of the other micronutrients of crp . although figure 1(b ) shows differences in the age profile of crp levels , the statistical analyses show that after allowing for other variables such as sex and for example , age , there are no significant differences in crp levels between the 2 age groups . overall , there was a significant positive association between rbp and levels of parasite - specific il-10 ( p = 0.049 , = 0.314 ) as well as between ferritin and parasite - specific il-4 ( p = 0.035 , = 0.317 ) . in some cases , the relationship between the cytokine levels and micronutrients varied with schistosome infection status as shown in table 1 . thus , levels of vitamin d showed a significant negative correlation with il-4 in egg positive children but no association in egg negative children ( figure 2(a ) ) . levels of parasite - specific ifn- showed a significant positive correlation with stfr in egg negative people but a negative but nonsignificant association in egg positive people ( figure 2(b ) ) . in egg positive people levels of parasite - specific il-5 went down with ferritin levels but went up in egg positive people although this later association was not significant ( figure 2(c ) ) . when considering the ratio of stfr , levels of both ifn- and il-4 went down with the stfr - f index in egg positive people and up in egg negative people as shown in figures 2(d ) and 2(e ) . this study describes the micronutrient status of a rural black zimbabwean population and then characterises the relationships between micronutrients and immune responses to schistosomiasis . while this study showed that there was vitamin d deficiency in the population , levels of all other micronutrients and markers of inflammation were within normal ranges . the global micronutrient report in 2001 has classified zimbabwe as having a vitamin a deficiency prevalence of 1015% . the study population had easy access to good dietary sources of micronutrients , including fortified foods ( margarine and some vegetable oils during the study period were fortified with vita ) as well as from home - grown vegetables . iron supplementation for pregnant women at ante - natal clinics and targeted vitamin a supplementation were not commenced in zimbabwe until 2 years after this current study was conducted . in this study serum retinol levels declined with age which is contradictory to reports from primary aged school children in zimbabwe and kenya [ 47 , 48 ] which show retinol levels increasing with age . work on rbp levels in exercise programs in south korean women revealed a larger decrease in older women than younger women after a structured exercise regime . this is consistent with our finding that rbp decreased with age , since our study captures a wider age range than the 2 previous studies in primary school children . however , the major occupation amongst our population is subsistence farming and so they are likely to be more physically active , therefore it is not clear whether our observations represent a normal decline in rbp with age , or whether there is an interaction between physical activity , age , and rbp level . found no association between s. haematobium infections with serum retinol levels in zimbabwe , similar to observations in this current study . interestingly , friis et al . found , a strong negative association between s. mansoni infection and serum retinol levels in both zimbabwe and kenya , which suggests that the intestinal niche of s. mansoni infection may interfere with vitamin a absorption [ 47 , 48 ] . however , experimental studies show that vitamin a deficiency leads to reduced schistosome - specific antibody responses , which may suggest that vitamin a deficiecy leads to susceptibility to s. mansoni infections . however , all participants of our study were negative for s. mansoni and therefore it was excluded as confounding factor . it has also been shown that all trans retinoic acid ( atra ) binds retinoic acid receptors , which induce foxp3 expression polarizing immune responses towards a regulatory phenotype . our finding that rbp is correlated with il-10 suggests that vitamin a may be important in augmenting schistosome - specific regulatory responses . vitamin d produced the most surprising data , with 38.5% of subjects being severely deficient . there is a paucity of vitamin d surveys in africa compared to those conducted in western countries . since no clinical examination were conducted in this study , it is impossible to say whether the deficiencies observed in this study results are associated with pathology or remained asymptomatic . production of pre - vitamin d3 occurs in the skin under the influence of ultraviolet light . most studies of vitamin d levels have been in caucasian populations with reference to osteoporosis . it is possible that our findings may be explained by ethnic differences in skin pigmentation and skin uv penetration [ 41 , 51 ] . given that the reference ranges come from studies on osteoporosis , they may not be applicable in zimbabwean population . nonetheless , they remain an important starting point for analysis and suggest that further work is required to examine the biological relevance of these categories to immunology . in this study , vitamin d levels in egg negative children showed a significant positive association with il-4 levels , consistent with the role of vitamin d in upregulating il-4 to polarize responses towards a th2 phenotype . iron deficiency is one of the most prevalent micronutrient deficiencies in the world affecting at least half of all pregnant women and young children in developing countries . in a survey conducted by the ministry of health and child welfare in 1997 , 9% of the surveyed population ( pregnant women , lactating women , preschool children , and adult males ) had depleted iron stores that is , ferritin . at the time of the study , pregnant and postpartum women were not offered iron supplementation by local healthcare providers , thus pregnancy and childbirth - related iron and blood loss may explain why male participants have significantly higher levels of ferritin . in this study , while ferritin levels were within normal ranges , stfr levels were elevated in 28.6% of the population . ferritin is an indicator of stored iron reserves in the body while stfr indicates the functional iron component of the body and becomes elevated soon after the onset of iron deficiency . ferritin is often decreased in iron deficiency anaemia , but can be raised in inflammatory conditions [ 27 , 39 ] . however , we observed normal crp levels , which excluded excess inflammation in the participants . similarly the lack of association between schistosome infection intensity / status and levels of stfr implies that schistosome infection does not explain the elevated levels of stfr . in this population the measures of body iron ( stfr - f index ) showed a negative association with ifn- and il-4 in egg positive people , while il-5 levels showed a positive association with ferritin in the same people . iron supplementation has been shown to increase dendritic cell stimulation and promote th1 responses , but also an increased burden of immunopathology in those already infected . however , increased ifn- is seen in iron deficiency , where it has a role in preserving iron stores [ 27 , 28 ] . thus in this population the inverse association between measures of body iron and the cytokines ifn- and il-4 may be adaptive to preserving iron stores during schistosome infection . the study showed that while levels of vitamin a and iron where within normal ranges , there was a deficiency of vitamin d in 67.2% of the study population as well as elevated levels of stfr in 28.6% of the participants . thus , the 2 indicators of iron levels suggested that although levels of stored iron were within normal levels ( normal ferritin levels ) , levels of functional iron ( measure by stfr ) may have been reduced in some participants . schistosome infection intensity or status was not associated with levels of any of the micronutrients , but altered the relationship between parasite - specific il-4 and il-5 and the measures of iron levels ( ferritin and stfr ) . cohort studies following a larger group of people through a cycle of antihelminthic treatment will clarify the effects of helminth infection on micronutrient levels and their subsequent effect on immune responses .

the association between hypertensive encephalopathy and cortical blindness in children with acute glomerulonephritis is extremely rare . we report the case of a 9-year old girl who presented with headache , seizures , altered sensorium , hematuria , and transient cortical blindness as a complication of hypertensive encephalopathy which showed complete reversal following normalization of blood pressure and an underlying post - infectious acute glomerulonephritis was revealed .

posterior reversible encephalopathy syndrome ( pres ) refers to a clinico - radiological entity characterized by headache , confusion , visual disturbances , seizures , and posterior transient changes on neuroimaging . we report a child with pres revealing post - streptococcal acute glomerulonephritis ( psgn ) . a 9-year - old girl presented to the er with history of low - grade fever for 7 days , sudden onset headache for 2 days , altered sensorium and three episodes of tonic clonic seizures since the previous day and loss of vision over the past 12 h. on examination , she was afebrile , irritable with altered sensorium , and mild puffiness of face . her blood pressure ( bp ) was 136/100 mm hg ( > 95 percentile for her age ) . she had no other focal neurological deficits and had no signs of any meningeal irritation . she was started on anti - hypertensive ( sublingual nifedepine ) , fluid and salt restriction , anti - edema measures , anticonvulsants , and other supportive care . investigations revealed hemoglobin 10.6 gm / dl , total count 18 500 cells / mm ( polymorphs 87% , lymphocytes 10% ) , platelets 404 000 cells / mm ; esr 9 mm ; c - reactive protein < 0.6 mg / dl ; raised blood urea nitrogen 40 mg / dl ; and creatinine 0.65 mg / dl . her serum electrolytes and liver function tests were normal . the urine collected after admission was cola - colored and urinalysis revealed plenty of rbcs with albuminuria 2 + . computed tomography ( ct ) of brain revealed symmetric occipital hypodense lesions with cerebral edema [ figure 1 ] . anti - streptolysin - o titer was positive ( 571 iu / ml ) and complement c3 level was low ( 40 mg / dl ) . computed tomography of brain at admission showing areas of hypodensity in the bilateral occipital lobes white matter with mild edema her bp gradually returned to normal range , and she slowly regained vision and sensorium within 10 h of hospitalization . imaging appearances and complete restoration of visual function following normalization of bp , a diagnosis of pres with psgn was made . follow - up ct scan 2 weeks after the first study showed complete resolution of the previous abnormal lesions [ figure 2 ] on a 1-year periodic follow - up in our unit , the child had no further recurrence of symptoms . follow - up ct scan of brain 2 weeks after the first study revealed complete resolution of the white matter abnormality in the occipital lobes posterior reversible encephalopathy syndrome , also known as reversible posterior leukoencephalopathy syndrome ( rpls ) was initially described by hinchey et al . in 1996 . it is a clinico - radiological entity characterized by neurological signs ( headache , seizures , vomiting , altered mental status , visual disturbances especially cortical blindness , focal neurological deficits ) and radiological abnormalities of occipital white matter , usually bilateral , characterized by cerebral edema with hypodense signals on ct scan ; and hyperintense signals on t2 weighted images by mri . the most common presenting symptoms were headache , seizure , altered consciousness , and cortical blindness . hypertensive encephalopathy , pre - eclampsia , eclampsia , systemic lupus erythematosus , wegener granulomatosis , minimal change nephrotic syndrome , chronic renal failure , post - transplantation , hemolytic uremic syndrome , acute intermittent porphyria , thrombotic thrombocytopenic purpura , vasculitis , malignancies , hypercalcemia , oxybutynin , intravenous immunoglobulins , organ transplantation , certain immunosuppressive , and cytotoxic drugs are among the known conditions associated with pres . two possible mechanisms proposed in the pathophysiology of pres are : ( i ) vasospasm due to acutely increased bp and ( ii ) loss of autoregulation . in the first hypothesis , it has been suggested that vasospasm contributes to ischemia and cytotoxic edema at regions of the arterial border zone . the second , more recent hypothesis is supported by diffusion images suggesting that dilation develops in cerebral arterioles due to autoregulatory failure . the objective of cerebral autoregulation is to keep blood flow constant , and to protect the brain during changes in bp ; however , sudden and severe increases in bp can impair autoregulation , and lead to arteriolar vasodilatation and endothelial dysfunction ( breakdown of blood brain barrier ) leading to extravasations of plasma and red blood cells causing vasogenic edema . the lesions of pres have a predilection for the parieto - occipital region , because the posterior cerebral arterial circulation supplied by the vertibro - basilar system has a lower level of sympathetic innervation and , therefore , is frequently involved . the role of neuro - imaging is to establish the initial diagnosis and to exclude other causes of neurological symptoms and signs . in pres , noncontrast ct ( ncct ) is sufficient to make the diagnosis in a proper clinical setting , revealing areas of low attenuation in posterior white matter . on mri brain , bilateral symmetrical edema in the parieto - occipital region is hyperintense on t2-weighted and fluid - attenuated inversion recovery ( flair ) sequences , and hypointense on t1-weighted sequences . diffusion - weighted imaging ( dwi ) can differentiate this condition from other major diseases such as infarction that are diffusion restricted . pres occurs most commonly in the setting of known hypertension or use of immunosuppressive agents . though the association between psgn and pres has been reported earlier,[1012 ] pres revealing acute glomerulonephritis is extremely rare in children . in our case , the girl presented with neurological symptoms , was found to be hypertensive , later developed gross hematuria after admission , and subsequently diagnosed with acute glomerulonephritis . our case highlights the possibility that cortical blindness may develop as a complication of acute glomerulonephritis in children . prevention of the occurrence of neurological deficits in children with acute glomerulonephritis and hypertensive encephalopathy requires careful evaluation and appropriate management of hypertension . in conclusion , it is important to consider this diagnosis in children presenting with encephalopathy and seizures in an appropriate clinical settings . pres is a treatable and reversible cause of acute encephalopathy with blindness , as long as an early diagnosis and appropriate treatment is made . without prompt treatment , the syndrome may lead to permanent brain injury or neurological sequelae such as chronic epilepsy . we need to be aware of this unusual neurological complication , as early recognition may improve prognosis .

in this review , the aspects and further developments of the concept of homeostasis are discussed also in the perspective of their possible impact in the clinical practice , particularly as far as psychic homeostasis is concerned . a brief historical survey and comments on the concept of homeostasis and allostasis are presented to introduce our proposal that is based on the classical assumption of the interstitial fluid ( isf ) as the internal medium for multicellular organisms . however , the new concept of a hierarchic role of isf of the various organs is introduced . additionally , it is suggested that particularly for some chemico - physical parameters , oscillatory rhythms within their proper set - ranges should be considered a fundamental component of homeostasis . against this background , we propose that the brain isf has the highest hierarchic role in human beings , providing the optimal environment , not simply for brain cell survival , but also for brain complex functions and the oscillatory rhythms of some parameters , such as cerebrospinal fluid sodium and brain isf pressure waves , which may play a crucial role in brain physio - pathological states . thus , according to this proposal , the brain isf represents the real internal medium since the maintenance of its dynamic intra - set - range homeostasis is the main factor for a free and independent life of higher vertebrates . furthermore , the evolutionary links between brain and kidney and their synergistic role in h2o / na balance and brain temperature control are discussed . finally , it is surmised that these two interrelated parameters have deep effects on the central nervous system ( cns ) higher integrative actions such those linked to psychic homeostasis .

homeostasis is a fundamental concept in physiology ; hence , it is a subject of the greatest interest for medicine ( 13 ) . in the present review , some aspects of the concept and possible further developments that could have an impact also in the clinical practice are analysed . our proposal is based on the classical assumption of the interstitial fluid ( isf ) as the internal medium for multicellular organisms . against this background , a central aspect is discussed ; namely the concept of the hierarchic role of the isf of the various organs in triggering homeostatic responses , especially of those involving overriding controls . in particular , we propose that the brain isf has the highest hierarchic role in human beings , providing the optimal environment for brain cell survival and complex functions , hence allowing the central nervous system ( cns ) to carry out its complex whole - body integrative actions , i.e. , to operate as the uppermost controller that allows a free and independent life of higher vertebrates . against this background , the crucial role of the kidneys for internal medium homeostasis will be revised according to the proposal that homer smith put forward more than fifty years ago in an extraordinary book ( 4 ) . as a matter of fact , homer smith pointed out , on the basis of evolutionary observations , that the functional interconnections between the brain and kidneys in the internal medium homeostasis are a basic requisite for a free and independent life of higher vertebrates . hence , it can be surmised that cns operates as the uppermost controller of the internal medium homeostasis not exclusively but especially thanks to its synergistic interactions with the kidney . the clinical evidence supports such a view as discussed below and reported in recent studies ( 5,6 ) . in particular , in this review , the brain / kidney synergistic roles in h2o / na balance and brain temperature control is discussed . actually , data are presented supporting the view that these two parameters are interrelated and have deep effects on the cns higher integrative actions . furthermore , the concept of allostasis , hence of a set - range of optimal values for internal medium parameters is assumed ( 7 ) . this view is further developed by considering the possible functional meaning of the rhythmic oscillations of the chemico - physical parameters inside their set - range that , according to our proposal , should be considered as a basic component of the homeostatic control . thus , the present proposal suggests that isf represents the real internal medium , since the maintenance of its homeostasis and in several instances the appropriate rhythms of some chemico - physical parameters within their appropriate set - range is the main factor for a free and independent life of higher vertebrates . in particular , the main focus of the present review is the suggestion that h2o / na balance and brain temperature play a crucial role in certain brain integrative actions . a special emphasis will be given to their rhythmic oscillations within their set - range that can have important modulatory actions on psychic homeostasis . as a matter of fact , psychic homeostasis ( see section below entitled ' body and psychic homeostasis ' for a definition ) refers to complex behavioural and bodily responses optimizing , inter alia , the interactions of a human subject with his socio - cultural environment ( i.e. , the so - called supra - systems ) . it should be underlined that psychic homeostasis can sometimes override bodily homeostasis regulatory mechanisms , i.e. , the controls aimed to optimize human being interactions with his external physical environment ( 7,8 ) . the need to characterize the complexity of the human being has led to a distinction between two elements : the soma and psyche ( 9 ) . thus , it has been proposed that two highly complementary homeostatic control processes are in operation in human beings : i ) the somatic physiological homeostasis , which via the integrative actions of the different apparatuses is capable of maintaining important chemico - physical parameters of the internal milieu within optimal set - ranges , allowing a free and independent life to the organism ; and ii ) the psychic homeostasis , which is aimed to an equilibrated mental condition via a proper resetting action of the different instances of the inner world and social environment that the subject has to harmonize . in agreement with such a view , freud proposed the principle of ' psychic homeostasis ' as a basic concept supporting his meta - psychology in his famous book ' the interpretation of dreams ' ( 10 ) . to indicate the psychic homeostasis in its proper set - range ( corresponding to a psychic state of subjective well - being ) the term eudaimonia ( also mentioned as eudaemonia ) , that consists of the words ' eu ' ( ' good ' ) and ' daimn ' ( ' spirit ' ) , has been proposed . thus , it could be described as a condition of dynamic balance between different appeals within the psychic sphere of the subject also in front of challenges of the social and physical environment . these aspects were further expanded ( 11 ) into the concept of ' predictive psychic homeostasis ' that designates the capability to create ( via the integrated actions of various brain - body mechanisms ) a psycho - physical state of readiness that anticipates potential threats . thus , predictive psychic homeostasis by avoiding possible future allostatic psycho - physical overloads is of a basic relevance for both eudaimonia and internal milieu homeostasis ( 8,11 ) . the survival value of predictive psychic homeostasis is emphasised by the evidence that it can sometimes override bodily homeostasis regulatory mechanisms ( 7,8 ) . it should be noted that both body homeostasis and psychic homeostasis are often jointly conditioned by the pattern of endocrine signaling ( 11,12 ) . in particular , alterations of anterior pituitary hormones leading to somatic disturbances have been demonstrated in stress and mood disorders , including major depression ( 13 ) . thus , hypothalamic - pituitary - adrenocortical hormones have deep effects on both body homeostasis and psychic homeostasis and it is interesting to point out that both are modulated by circadian secretion rhythms ( 14,15 ) . in other words , human cns high integrative actions link together human basic physiology with the subject 's supra - systems that play a basic role in psychic homeostasis . thus , for the human being psychic homeostasis and bodily homeostasis are two inseparable aspects of his health . such a view reminds aphorisms of ancient medicine and philosophy that have proposed the soul serenity ( eudaimonia , i.e. , the good spirit ; see also the discussion ) as the primary aim of subject 's well - being . obviously , eudaimonia is deeply dependent on gratifying interactions between the individual human being and his socio - cultural environment ( 16,17 ) . the need to characterize the complexity of the human being has led to a distinction between two elements : the soma and psyche ( 9 ) . thus , it has been proposed that two highly complementary homeostatic control processes are in operation in human beings : i ) the somatic physiological homeostasis , which via the integrative actions of the different apparatuses is capable of maintaining important chemico - physical parameters of the internal milieu within optimal set - ranges , allowing a free and independent life to the organism ; and ii ) the psychic homeostasis , which is aimed to an equilibrated mental condition via a proper resetting action of the different instances of the inner world and social environment that the subject has to harmonize . in agreement with such a view , freud proposed the principle of ' psychic homeostasis ' as a basic concept supporting his meta - psychology in his famous book ' the interpretation of dreams ' ( 10 ) . to indicate the psychic homeostasis in its proper set - range ( corresponding to a psychic state of subjective well - being ) the term eudaimonia ( also mentioned as eudaemonia ) , that consists of the words ' eu ' ( ' good ' ) and ' daimn ' ( ' spirit ' ) , has been proposed . thus , it could be described as a condition of dynamic balance between different appeals within the psychic sphere of the subject also in front of challenges of the social and physical environment . these aspects were further expanded ( 11 ) into the concept of ' predictive psychic homeostasis ' that designates the capability to create ( via the integrated actions of various brain - body mechanisms ) a psycho - physical state of readiness that anticipates potential threats . thus , predictive psychic homeostasis by avoiding possible future allostatic psycho - physical overloads is of a basic relevance for both eudaimonia and internal milieu homeostasis ( 8,11 ) . the survival value of predictive psychic homeostasis is emphasised by the evidence that it can sometimes override bodily homeostasis regulatory mechanisms ( 7,8 ) . it should be noted that both body homeostasis and psychic homeostasis are often jointly conditioned by the pattern of endocrine signaling ( 11,12 ) . in particular , alterations of anterior pituitary hormones leading to somatic disturbances have been demonstrated in stress and mood disorders , including major depression ( 13 ) . thus , hypothalamic - pituitary - adrenocortical hormones have deep effects on both body homeostasis and psychic homeostasis and it is interesting to point out that both are modulated by circadian secretion rhythms ( 14,15 ) . in other words , human cns high integrative actions link together human basic physiology with the subject 's supra - systems that play a basic role in psychic homeostasis . thus , for the human being psychic homeostasis and bodily homeostasis are two inseparable aspects of his health . such a view reminds aphorisms of ancient medicine and philosophy that have proposed the soul serenity ( eudaimonia , i.e. , the good spirit ; see also the discussion ) as the primary aim of subject 's well - being . obviously , eudaimonia is deeply dependent on gratifying interactions between the individual human being and his socio - cultural environment ( 16,17 ) . the french physiologist claude bernard ( 18131878 ) introduced the concept of the internal environment of an organism ( 18 ) , upon which walter cannon ( 18711945 ) proposed the concept of homeostasis . a crucial aspect is obviously the definition of internal medium and the characterization of its distinctive features in physiological terms ( 19 ) . initially bernard postulated that blood was the milieu interieur , or internal environment , exchanging substances and operating as an optimal environment for cells metabolism of higher animals . thus , he proposed the concept of the fixity or constancy of the internal environment as essential to the vital processes by stating : ' the fixity of the milieu supposes a perfection of the organism such that the external variations are at each instant compensated for and equilibrated . the stability of the internal environment is the condition for the free and independent life ' ( 20 ) . later on the interstitial fluid , as the environment with which the cells of a multicellular organism exchange , was proposed and the concept of ' homeostasis ' was introduced by cannon . the new term was defined as follows ( 21 ) : ' the constant conditions which are maintained in the body might be termed equilibria . that word , however , has come to have fairly exact meaning as applied to relatively simple physico - chemical states , in closed systems , where known forces are balanced . the coordinated physiological processes which maintain most of the steady states in the organism are so complex and so peculiar to living beings - involving , as they may , the brain and nerves , the heart , lungs , kidneys and spleen , all working cooperatively - that i have suggested a special designation for these states , homeostasis . it means a condition - a condition which may vary , but which is relatively constant . homeostasis , at least in the narrow sense , is about preserving the status quo , regulation to achieve viability through resistance to change . ' furthermore , cannon adapted the idea of homeostasis to the organism as a whole , hence referring to both physiological and psychological aspects of control mechanisms . as mentioned above , this broadening of the homeostasis concept to the psychic aspects is of peculiar relevance for the human being and it will be considered in the present review . let us discuss with some detail the concept of allostasis that was introduced in 1988 by sterling ( 7 ) , as a further development of some aspects of the concept of homeostasis ( 22 ) . in fact , allostasis points out that the living systems are endowed with the adaptability property , hence the control mechanisms operate for a continuous adjusting to change , not simply by restoring , but rather by altering equilibrium settings in order to face in an optimal fashion the new environmental inputs . thus , not a set - point for the controlled parameter but rather a set - range is maintained ( fig . , allostasis describes the processes through which organisms actively maintain a dynamic steady state of conditions for basic parameters necessary not simply for their survival but rather for optimal interactions with the environment . it should be underlined that allostatic mechanisms can also adapt the organisms to probable future loads ( bayesian approach ) and this is obviously especially true for organisms that are endowed with a complex cns . thus , the concept of allostasis was introduced to underline both physiological and psychological change and adaptation to diverse present and surmised future circumstances . obviously , the behavioural and physiological anticipatory responses to probable future events emphasize the crucial role of a complex cns , particularly for responses allowing the so - called predictive allostasis ( 1,78,16,2326 ) . we should also mention another far - sighted proposal of cannon , since he underlined that the internal environment with which the cells exchange is not simply a watery environment but rather a ' fluid matrix ' whose chemical and also physical characteristics , such as temperature and pressure , are of a basic importance . it should be noted that the fluid matrix [ hence the aqueous extracellular matrix ( ecm ) ] forming the internal medium is the product of the organism itself and it can play a basic role in the intercellular communication processes ( 2730 ) . the evidence that the internal medium and in particular the ecm is produced by the organism according to its functional needs implies a broader view of homeostasis , since the self - construction of the internal medium is in some way related to concepts introduced in the 1970s by varela et al ( 31 ) and discussed by others by stating ( 3133 ) : ' a living system is a system capable of self - reproduction and self - maintenance through a regenerative network of processes which take place within a boundary of its own making and regenerates itself through cognitive or adaptive interactions with the medium ' ( 33 ) . thus , a living system can be characterized by two notions ( 3335 ) : i ) the first one is ' autopoiesis ' , a term indicating the ability of the system to transform external matter / energy into an internal process of self - maintenance and production of its own components . autopoiesis , therefore , is concerned with internal structure and describes a system organized as a network of interactions capable of cyclically reproducing the components to allow the system 's self - perpetuation ; and ii ) the second one can be called ' cognition ' to indicate that the interaction with the environment is implemented on the basis of the internal logic of the living system . the environment , in other words , does not prescribe or determine changes to the organism , it is the structure of the living system and its previous history of perturbations that determine what reactions a new perturbation will induce . cognition , therefore , concerns the relationship of the system with the environment . it describes a system able to sense the external world in order to produce a response to perturbations either by self - correction of its disturbed organization ( homeostasis ) , or by reorganizing itself into a different self - perpetuating network of interactions ( adaptability ) . there are , of course , various levels of cognition ( corresponding to different levels of life 's complexity ) from very simple mechanisms of cell sentience ( 36 ) up - to complex functions performed by nervous systems . this view captures the essence of cellular life by recognizing that life is a cyclic process that produces the components that in turn self - organize in the process itself , and all within a boundary of its own making . such a self - constructed machine continuously operates to maintain critical chemico - physical parameters within a suitable set - range in spite of perturbing influences . as pointed out by wiener in his classical book ( 37 ) , the mechanisms allowing the processes of maintenance of the values of critical parameters within suitable set - ranges that allow survival of the living beings notwithstanding the external environment changes can be described according to cybernetic models . during the middle of the past century , norbert wiener ( 18941964 ) pointed out that the concept of homeostasis was common to animals and to automatic systems ( 37 ) . thus , he defined cybernetics as the entire field of the theory of communication and control in the living beings and in the automatic systems . during the 1940 's and 1950 's , cybernetics had a crucial influence on the birth of various modern sciences : control theory , computer science , information theory , automata theory , artificial intelligence and artificial neural networks , cognitive science , computer modelling and simulation science , dynamical systems , and artificial life . however , after these disciplines had become fully independent , cyberneticists felt the need to further develop their field to distinguish an approach emphasizing autonomy , self - organization , cognition from more mechanistic control strategies followed in control engineering and computer science . in the early 1970 's , this effort became known as ' second - order cybernetics ' ( 38 ) and provided a thoughtful revision of concepts of particular relevance for biology and physiology . in particular , the negative feedback ( fb ) is a type of control central to both homeostasis and cybernetics , since it can explain how a living being or an automatic system can operate for maintaining its steady state in spite of disrupting influences from the external environment ( fig . 1 ) . the fb control can be described as follows : let us suppose that a stress from the external environment acts upon a system of an organism and such a system is under a fb control . a sensor detects the stress signal and converts it into signals that are meaningful for the internal control mechanisms of the organism . a controller compares the signals to the desired set - points , and issues error signals if there are discrepancies . thus , each error signal refers to the difference between the optimal value of a variable ( set - point ) and the value returned by the homeostatic mechanism(s ) during the persisting perturbation . the error is interpreted by an activator , which triggers the appropriate response of effectors to restore the value of the variable close to its set - point or , according a more realistic view , within a suitable set - range ( fig . as pointed out above , in a living being , several regulatory mechanisms are continuously activated to produce the homeostatic responses ; hence , in analyzing the allostatic controls the following aspects should at least be considered : i ) redundancy and inter - dependence , since several negative feedbacks are directly or indirectly involved in the control of the same functional parameter or of functional parameters strictly interdependent ( e.g. , blood volume and arterial blood pressure ) ; ii ) push - pull controls ( e.g. , glucagon / insulin ) allowing a precise control of the parameter ; and iii ) russian doll organization of controls of increasing miniaturization ( 3941 ) . thus , it is possible to recognize fb control at the systems ( set of apparatuses ) level , at the level of organs , at the level of tissues , at the level of the cells , at the level of the molecular networks . the crucial role for overriding controls is played by the cns via its interactions with the peripheral apparatuses especially for parameters , such as arterial blood pressure , involved in facing challenging conditions ( e.g. , in fight or flight conditions ) ( 4248 ) . taken together , it can be stated that homeostasis implies the regulated activation of several dynamic and potentially predictive multilevel processes organized according to a russian doll pattern ( 32,42,43,49,50 ) . furthermore , as proposed by ' brain - body medicine ' , the cns plays a crucial role in homeostasis by interacting with practically all the peripheral organs ( 42,5153 ) . in this context , the brain / kidney interactions for the h2o / na and brain temperature control that are interrelated parameters of basic importance for cns integrative actions , will be discussed . as discussed below , such an assumption can also be surmised by deductions based on the evolutionary background . during the middle of the past century , norbert wiener ( 18941964 ) pointed out that the concept of homeostasis was common to animals and to automatic systems ( 37 ) . thus , he defined cybernetics as the entire field of the theory of communication and control in the living beings and in the automatic systems . during the 1940 's and 1950 's , cybernetics had a crucial influence on the birth of various modern sciences : control theory , computer science , information theory , automata theory , artificial intelligence and artificial neural networks , cognitive science , computer modelling and simulation science , dynamical systems , and artificial life . however , after these disciplines had become fully independent , cyberneticists felt the need to further develop their field to distinguish an approach emphasizing autonomy , self - organization , cognition from more mechanistic control strategies followed in control engineering and computer science . in the early 1970 's , this effort became known as ' second - order cybernetics ' ( 38 ) and provided a thoughtful revision of concepts of particular relevance for biology and physiology . in particular , the negative feedback ( fb ) is a type of control central to both homeostasis and cybernetics , since it can explain how a living being or an automatic system can operate for maintaining its steady state in spite of disrupting influences from the external environment ( fig . 1 ) . the fb control can be described as follows : let us suppose that a stress from the external environment acts upon a system of an organism and such a system is under a fb control . a sensor detects the stress signal and converts it into signals that are meaningful for the internal control mechanisms of the organism . a controller compares the signals to the desired set - points , and issues error signals if there are discrepancies . thus , each error signal refers to the difference between the optimal value of a variable ( set - point ) and the value returned by the homeostatic mechanism(s ) during the persisting perturbation . the error is interpreted by an activator , which triggers the appropriate response of effectors to restore the value of the variable close to its set - point or , according a more realistic view , within a suitable set - range ( fig . 1 ) . as pointed out above , in a living being , several regulatory mechanisms are continuously activated to produce the homeostatic responses ; hence , in analyzing the allostatic controls the following aspects should at least be considered : i ) redundancy and inter - dependence , since several negative feedbacks are directly or indirectly involved in the control of the same functional parameter or of functional parameters strictly interdependent ( e.g. , blood volume and arterial blood pressure ) ; ii ) push - pull controls ( e.g. , glucagon / insulin ) allowing a precise control of the parameter ; and iii ) russian doll organization of controls of increasing miniaturization ( 3941 ) . thus , it is possible to recognize fb control at the systems ( set of apparatuses ) level , at the level of organs , at the level of tissues , at the level of the cells , at the level of the molecular networks . the crucial role for overriding controls is played by the cns via its interactions with the peripheral apparatuses especially for parameters , such as arterial blood pressure , involved in facing challenging conditions ( e.g. , in fight or flight conditions ) ( 4248 ) . taken together , it can be stated that homeostasis implies the regulated activation of several dynamic and potentially predictive multilevel processes organized according to a russian doll pattern ( 32,42,43,49,50 ) . furthermore , as proposed by ' brain - body medicine ' , the cns plays a crucial role in homeostasis by interacting with practically all the peripheral organs ( 42,5153 ) . in this context , the brain / kidney interactions for the h2o / na and brain temperature control that are interrelated parameters of basic importance for cns integrative actions , will be discussed . as discussed below , such an assumption can also be surmised by deductions based on the evolutionary background . as stated in the general premise , it is proposed that the brain isf plays the highest hierarchic role among the isf of the different organs of the human body in triggering homeostatic responses of fundamental importance . such a strong assumption does not deny the relevance of the holistic view of the human body as proposed by ' brain - body medicine ' ( 42 ) , but it is aimed at suggesting a frame to organize the data indicating that the isf dyshomeostasis reduces to a great extent the cns integrative actions that allow a free and independent life . as mentioned above , to support our assumption , we focus our analysis on two parameters , namely the h2o / na and brain temperature control and the cns / kidney synergistic interactions . as an introduction to this subject , a preliminary analysis is proposed on the complex fluid and cellular movements among the various brain fluid compartments , since they are involved not only in the metabolic support to neurons and glial cells and in the clearance of waste molecules from the brain , but also in the brain interactions with the systemic apparatuses . in an interesting study , brinker et al published a very useful diagram showing the complex fluid and cellular exchanges between the brain fluid compartments ( 54 ) and we will refer to it for our proposal . actually , according to our view , it is possible to organize the brain fluid compartments distinguishing a ' major cerebrospinal fluid ( csf ) circulation ' ( black arrows in fig . 2 ) from an ' inter - compartments fluid circulation ' ( blue arrows in fig . 2 ) from a ' csf intra - ventricular local circulation ' characterized by gentle ebbs ( broken arrows in fig . 2 ) . the schematic representation suggests a special hub status for isf in view of its multiple connections with the other fluid compartments . furthermore , a privileged status for isf is pointed out by the limiting barriers that regulate its cellular and fluid exchanges with the other compartments . actually , isf is protected by a highly selective barrier from the cerebral blood circulation and by a permeable barrier formed by gap junctions in the pia and ependymal layer and by the brain tissue microarchitecture ( e.g. , tortuosity and viscosity of the brain ecm ) from csf . furthermore , it should also be pointed out that isf and virchow - robin space ( vrs ) are separated only by the glial barrier through which also cells can move to isf . as indicated in fig . 2 , csf operates as an interface between the major and minor circles . in addition , csf exchanges signals with isf not only directly but also indirectly via its reciprocal interactions with the vrs . thus , csf secretion , composition and circulation have a central importance for isf homeostasis . as far as the secretion of csf is concerned , the choroid plexus ( cp ) has been proposed as the main locus for the secretion and clearance of csf . thus , at cp the blood - csf barrier secretes and purifies csf , generates intracranial pressure and maintains the csf composition . therefore , the cp secretory epithelium plays a basic role for brain homeostasis and integrative actions via multiple transport and secretory functions that support csf homeostasis and its fluid dynamics ( 55 ) . as far as csf composition is concerned , it is important to compare at least some of its main chemico - physical parameters with those of plasma and extracellular fluid ( ecf ) . two highly selective barriers separate csf and isf from the blood , while they are separated from each other by easily permeable boundaries . thus , if isf has the highest hierarchic order among the brain isf , csf should be also considered in view of its continuous renewal and circulation as a crucial fluid compartment for brain integrative functions and as a good mirror of brain isf . on the other hand each of these will contribute to plasma composition according to its volume , composition and access to plasma . thus , brain isf also in view of the blood - brain barrier ( bbb ) will not greatly contribute to plasma composition notwithstanding its high hierarchic order . these aspects are illustrated in fig . 2 and in table i where some of the main chemico - physical parameters of plasma , ecf and csf are presented . as far as csf circulation i.e. , the ' third circulation ' according to cushing ( 56 ) is concerned , it has been shown that adult cp secretes csf that flows through the ventricles into the subarachnoid space , then over the top of the brain , with some csf flowing down to the lumbar sac . the exit of csf is mainly to the blood at the arachnoid granulations but also to the lymph system through the nasal cribriform plate or via the spinal nerve roots ( 55 ) . however , csf flow is not unidirectional since it has been shown that not only is a pulsatile flow but also characterized by up and down movements throughout the entire brain with local fluid exchanges with isf , circumventricular organs ( cvos ) and vrs . furthermore , it should be underlined that the csf has not only gentle and bidirectional ebbs and flows that create mixing ( 55 ) , but it has also been shown that the single layer of ciliated ependymocytes in contact with the csf beat in a periodic manner , generating localized microscale ( beating of cilia ) effects on near - wall csf dynamics ( fig . 2 ) . this action may contribute in clearing debris from the ventricle walls , and may enhance mixing , which may be particularly relevant for neuroendocrine communication ( 57 ) or even for signal detection ( 36 ) . thus , while some short - distance volume transmission ( vt)-signalling can rely upon this action of the ependymal cilia close to the walls of lateral ventricles , long - distance vt - signalling could be mainly driven by wall motion and cp pulsation , hence by csf dynamics in the centre regions of the ventricles , according to the tide hypothesis ( 58 ) . thus , it has been suggested to distinguish a major csf circulation that is the classical one from the cp to the arachnoid granulations , from a local csf micro - circulation that is mainly confined close to the ciliated walls of ventricles . while the former plays a role as nourishing liquor and long - distance pathway for vt signal migration , the latter one is mainly involved in short distance migration of vt signals and csf flow - mediated neuronal guidance ( 57,59,60 ) . furthermore , the csf circulation around blood vessels penetrates from the subarachnoid space into the vrs allowing cell exchange between blood and brain . as proposed by bechter et al ( 6062 ) , csf is also a diffusion pathway for pathogenic agents causing neuropsychiatric diseases ( 63 ) . actually , it has been shown that blood - borne inflammatory cells enter the brain via vrs or via cp and clinical evidence supports the relevance of an immunological crosstalk between periphery and intrathecal immunity in neurological and psychiatric diseases ( 54,64 ) . the fluid movements at the barriers are driven by osmotic and hydrostatic gradients or by active transporter processes . aquaporins and other transporters play a fundamental role in the control of fluid exchange at the glial , endothelial , and cp barrier . in particular , the water influx into the vrs and , hence , interstitial flow is regulated by aquaporin-4 localized in the end feet of astrocytes ( 65 ) . it is interesting to note that the fluid movements into and out of the vrs depend on respiratory and cardiac pressure pulsations and can be an important component of the tide hypothesis for vt signal diffusion in the brain ( 58 ) . a special role of tanycytes for brain - body interactions has also been proposed , since tanycyte - like cells are a characteristic feature of all cvos and they display well - organized tight junctions around their cell bodies that can represent a bbb displaced from the vascular to the ventricular side ( 66 ) . thus , tanycytes may play an important role in regulating the exchange between blood , csf and isf , and hence in regulating the brain - body interactions , particularly in some organs that are deeply involved in the control of internal medium as the kidney . the assumption of peculiar brain / kidney interactions finds an indirect support from comparative physiology and evolutionary studies . in the famous and fascinating book cited above , homer smith ( 4 ) proposed that the following processes do indicate a parallel evolution of the nervous system and kidneys ( fig . 3 ) : i ) the increased complexity of the renal apparatus has allowed a progressively more precise control of the volume and composition of the internal medium ; ii ) the precise control of the volume and composition of the internal medium has allowed the establishment of a progressively more complex nervous system ; and iii ) the organism endowed with a complex cns has become capable of multidimensional allostatic controls , i.e. , of complex dynamic predictive homeostasis , particularly for chemico - physical parameters that are highly relevant for cns integrative actions . accordingly , some aspects will be discussed as to how a complex cns operates to allow interconnections between the brain and kidneys for the homeostatic controls of sodium concentrations and brain temperature . as far as plasma sodium concentrations are concerned it should be noted that this parameter not only has a restrict set range ( 135 meq / l < [ na ] < 147 meq / l ) , but also that hyponatremia causes important cns symptoms ( e.g. , amnesias , headaches , epilepsy ) ( 5 ) . as far as cerebral temperature is concerned , it should be noted that brain deep structures are at 12c above the body core temperature . thus , blood in the cerebral circle is at a a lower temperature than the deep brain structures hence cerebral blood flow besides providing nutrients to the brain and allowing the clearance of its metabolic waste products has also the function of absorbing heat especially from the active brain regions ( 6770 ) . the osmotic regulation of water homeostasis and body core temperature are functionally related , since the maintenance of body water homeostasis is critical for preventing hyperthermia , as evaporative cooling is the most efficient means of dissipating excess body heat . as far as water homeostasis is concerned , its control is mainly achieved by the regulation of water intake and water loss by the kidneys . the former is achieved by sensations of thirst that motivate water intake , whereas the latter is regulated by the antidiuretic action of vasopressin . vasopressin ( also known as antidiuretic hormone ( adh ) ( 5 ) secretion and thirst are stimulated by increases in the osmolality of the ecf , as well as by decreases in blood pressure and/or blood volume , signals that are precipitated by water depletion associated with the excess evaporative water loss required to prevent hyperthermia . thus , it can be deduced that thermoregulation and osmotic regulation of water homeostasis are functionally interdependent . let us briefly mention two important recent contributions on the mechanisms involved in this integrated homeostatic control ( 71,72 ) . actually , adh enhances thermo - sensitivity of warm - sensitive neurons and decreases thermo - sensitivity of cold - sensitive neurons . such adh modulatory actions promote heat loss and are probably involved in the adh - induced hypothermia ( 71 ) . the basic role of adh for both osmotic regulation of water homeostasis and cerebral homoeothermic control are schematically summarized in fig . another mechanism involved in the integration of thermal and the osmotic regulation of water homeostasis has also been described , that is based on the transient receptor potential - vanilloid ( trpv ) family of ion channels that respond both to osmotic signals and also are stimulated by increases in body temperature ( 72 ) . as described in the recent study by noda et al ( 6 ) , at least two independent sensing systems in the cvos are involved in monitoring osmolality and body - fluid volume , namely na(x ) channels and trp channels . in particular , na(x ) channels have been demonstrated to play a role as [ na ] sensors and trp channels as osmosensors , but these channels are also temperature - sensitive . let us briefly describe these two molecular mechanisms involved in the control of na / h2o and cerebral temperature : i ) na(x ) channels are located in perineuronal processes and in the ependymal cells of the subfornical organ and organum vasculosum lamina terminalis ( ovlt ) and are activated by hypernatremia . their activation causes a reduction in na assumption ; and ii ) trpv channels , such as the trpv1 and trpv4 are expressed at the level of the supraoptic nucleus and at the level of the ovlt . these channels are activated by hyper - osmolality but also by increases in the cerebral temperature . in other words , there are brain regions ( particularly cvos ) and central molecular mechanisms ( e.g. , trpv ) allowing brain - kidney interactions in the control of ecf osmolality and cerebral temperature ( 72 ) . thus , apart from the mechanism that is dependent on adh modulatory actions on hypothalamic thermo - receptors and via such an action integrate thermal and osmotic regulation , the mechanism that is dependent on tprv channels whose activation is directly modulated by fluid osmolality and cerebral temperature should also be considered . the triggering of these mechanisms also depends on multiple neural and humoral signals from the periphery . the latter ones can reach the brain via the cerebral circulation and from the blood can reach the brain integrative centres , particulatly those located in the cvos , since they are outside the bbb ( 5466 ) . in the frame of the basic problem already faced by claude bernard , that is of the definition of internal medium and the characterization of its distinctive features in physiological terms , we propose that there is a hierarchic order among the isf and we maintain that brain isf is the real ' internal medium ' of higher vertebrates since it is the most important isf for triggering the complex responses allowing a free and independent life and this is particularly true for human beings . as mentioned above , this assumption does not deny the holistic view of the human organism but it gives a further aspect to the ' brain - body medicine ' that has recently been investigated also for a surprising new aspect , namely the synergistic role between brain and gut microbiota ( 73 ) . thus , while the general relevance of the brain / body interactions are a main subject of investigations the main highly focused subject of our paper is how brain circuits and kidney functions can modulate brain isf composition . thus , according to our proposal , brain isf represents the ' core fluid compartment ' that provides an optimal environment for development , survival and , together with csf , it allows vt intercellular communications among the brain complex cellular networks ( 58,74 ) . thus , brain isf is likely a crucial functional component of the multifaceted integrative cns actions that are involved in most of the dynamic predictive homeostatic mechanisms and overriding controls . the crucial role of brain isf is also indicated by the evidence that does exchange signals with the other brain fluid compartments but only in a highly regulated way ( fig . our view is that brain isf has the highest hierarchic role in triggering dynamic homeostatic responses involved in the balance of volume and sodium concentrations of the ecf as well as the cerebral temperature control . as discussed above , it is suggested that these two functionally interrelated parameters play a crucial role for the high level integrative actions of cns as those involved in the control of the psychic state . in agreement with such a view many clinical studies demonstrate correlations between the water / sodium balance , homoeothermic balance and psychic conditions of the subject . actually , it has been reported that : i ) psychic stresses increase the plasma and csf levels not only of cortisol but also of adh ( 75 ) ; ii ) adh plasma levels are increased in depressed patients ( 76 ) ; iii ) epidemiological studies have demonstrated a correlation between depression and habitats with a high salinity ( 77 ) ; iv ) schizophrenic patients have alterations in the control of body temperature ( 78 ) ; and v ) rpv1 channels beside responding to the osmotic signals are also activated by cerebral temperature ( 6,72 ) and at the level of the cerebral cortex are altered in depression ( 79 ) . it has been introduced the term of psycho - physic allostasis to indicate the responses aimed not only to the maintenance of the internal medium homeostasis but also to the broader condition of well - being of the subject hence to his eudaimonia ( 16,17 , see section ' body and psychic homeostasis ' ) . this condition of well - being especially depends on the interactions between the systems of the organism and the supra - systems . thus , eudaimonia does depend from the interactions between the human being and external physical environment but even more from the interactions of the subject with his socio - cultural environment . according to our proposal , the brain isf and its ' mirror fluid ' the csf have the highest hierarchic role since they allow optimal conditions for cns integrative actions . in particular , body - fluid and core temperature controls are two chemico - physical parameters that are essential for a free and independent life of human beings that is possible only if their cns operates in optimal conditions . furthermore , we have proposed that in several instances an appropriate oscillatory rhythm ( often but not exclusively a circadian rhythm ) of a chemico - physical parameter within its appropriate set - range is an essential component of allostasis . thus , in several instances the load error is the result of the discrepancy between the internal medium value and not simply the set - range values but the appropriate oscillatory rhythm that the chemico - physical parameter under scrutiny should have . a consequence of such an assumption is that dyshomeostasis can be actually present even if the chemico - physical parameter under scrutiny is within its appropriate set - range but with inappropriate rhythmic oscillations . thus , a careful analysis of chemico - physical parameters dynamic patterns should be considered and not only at plasmatic level but also at csf level . as a matter of fact , it is interesting observing that the concentration rhythms of sodium are differentially regulated ( fig . 5 ) in the csf ( 80 ) in comparisons with the sodium concentrations rhythms in the systemic circulation ( 81 ) . this aspect has clinical implications not only since it has been shown that a correlation exists between the rising levels of csf sodium levels and the timing of migraine onset ( 80 ) but also since as recently stated water and sodium homeostasis is inextricably coupled to the cns integrative actions ( 82 ) . in this regard , neuroscience describes an inverse relationship between the intensity of the neuropil function and the amount of water it contains with different shrinkage the ecm according to the neuronal activity ( 30,82,83 ) . as discussed above , these variations in the ecm perviousness have profound effects on vt signals diffusion ( 29,30,74 ) hence also regulate the exchange of signals isf / blood , isf / csf , isf / vrs . thus , exchanges between blood - born and brain - born vt signals depend also at some extent on the ecm hydration and composition ( 30,82,83 ) . this could be one important feature characterizing brain - kidney interactions since as mentioned above integrative centres for the feedback mechanisms involved in na / water and cerebral temperature homeostasis are localized at cvos level that is outside the bbb but separated by barriers from isf . against this background and on the bases of recently published data ( 6,72 ) it has been proposed that ( na ) , osmolality and cerebral temperature are continuously monitored in the brain in particular at cvos level and are strictly regulated thanks to brain - kidney interactions to maintain the optimal set - range for brain isf that , in turn , is a necessary prerequisite for the brain integrative actions . furthermore , on the basis of the clinical data above mentioned it has been proposed that ( na ) , osmolality and cerebral temperature are interconnected parameters playing a fundamental role in the psychic allostasis . thus , it is surmised that clinical evaluations of these parameters are of paramount importance for the psychic homeostasis of the subject and could be modulated by optimizing his interactions with the socio - cultural environment in which he lives as well as by psycho - therapy . in other words , it could be proposed the potential relevance of monitoring internal medium ( na ) and cerebral temperature during psycho - therapy treatments like the ones administrated to patients suffering of post - traumatic stress disorders ( 16,8486 ) . thus , it could be relevant to evaluate the physical ( internal medium parameters ) and psychological ( eudaimonia ) therapeutic effects produced by addressing the patient 's inner speech in such a way of favoring his predictive psychic allostasis to peaceful scenarios . this could lead to a better dealing with the stressing actions of supra - systems and the traumatic memory traces that he has to face ( 23,85,86 ) .

in stable critically ill children , the adoption of a restrictive transfusion strategy based on a predefined hemoglobin threshold of 7 g / dl significantly decreased transfusion requirements without affecting outcome . these results strengthen previous observations made in volume resuscitated adults when a similar blood transfusion strategy was used . it also indirectly corroborates studies reporting the beneficial effects of leukoreduction of red blood cell ( rbc ) transfusion units on patient outcome . this study indicated that the maintenance of a higher hemoglobin concentration with rbc transfusion in an attempt to increase tissue oxygen delivery is not associated with a clinical benefit . this may be related to the storage process , which could affect the ability of rbcs to transport and deliver oxygen to the tissues . this point , however , remains controversial . it should also be remembered that increasing hemoglobin concentration will not always result in a greater oxygen delivery , as transfusion related increased blood viscosity could be associated with a reduction in blood flow . further research should compare a symptomatic transfusion strategy to a hemoglobin - based strategy on the outcome of high risk patients .

the transfusion of red blood cell ( rbc ) concentrates in critically ill patients remains controversial and has generated much research and debate in the medical literature . a recent , large , noninferiority randomized clinical trial adds an important piece to this quite complicated ' puzzle ' . in stable critically ill anemic ( hemoglobin < 9.5 g / dl ) children between 3 days and 14 years of age , this study demonstrated that a restrictive strategy , where the threshold hemoglobin concentration was 7 g / dl , significantly decreased transfusion requirements without increasing adverse outcome , defined as a composite of death and development of new or progressive organ failure , when compared to a liberal strategy with a threshold hemoglobin of 9.5 g / dl . anemia is common in critically ill patients and results in a large number of rbc transfusions . several studies reported that up to 50% of adult or children who were hospitalized in an intensive care unit received rbc transfusions [ 2 - 4 ] . interestingly , all these observational studies reported that hemoglobin level , rather than clinical or physiological factors , drives transfusion decision . the adequacy of any hemoglobin concentration in a given clinical situation depends on whether a sufficient amount of oxygen is carried to the tissues to meet their metabolic requirements . the optimal hemoglobin threshold for rbc transfusion in different populations , and especially in critically ill patients , remains unknown . the study of lacroix and colleagues confirms the results reported by two other randomized trials that evaluated the impact of a restrictive strategy on the outcome of critically ill adults and preterm infants . using 30-day mortality as the primary outcome in 838 euvolemic adult critically ill patients , hbert and colleagues demonstrated that a restrictive transfusion strategy was at least as effective as a liberal one . in addition , applying a liberal transfusion strategy resulted in a significantly higher multiple organ dysfunction score , a composite outcome taking into account 30-day mortality and the number of organ failures . this deleterious effect might be attributed to the fact that rbc units transfused in this study were not leukocyte - reduced , in contrast to the rbc units used in the study by lacroix and colleagues . two ' before and after ' studies in adults and premature infants and one meta - analysis of randomized controlled trials have reported that leukoreduced rbc transfusion could significantly improve the outcome of high risk patients [ 8 - 10 ] . it has been decided , therefore , to repeat a prospective controlled randomized study to compare hemoglobin thresholds of 7 versus 9 g / dl . using a composite primary outcome including death before home discharge or survival with any of severe retinopathy , bronchopulmonary dysplasia or brain injury on cranial ultrasound in 451 infants with birth weight < 1,000 g , kirpalani and interestingly , the thresholds developed in this study were based on whether or not the infant was receiving respiratory support . although not specified , as the study was performed after 1999 and included canadian centers , it may be reasonably assumed that the authors used leukoreduced rbc units . the results of this study are in contrast with those of bell and colleagues , who reported in a smaller trial ( n = 100 ) that infants in the restrictive - transfusion group were more likely to have intraparenchymental brain hemorrhage or periventricular leukomalacia . however , this combination was not a pre - specified outcome and the study was powered for the primary outcome of number of transfusions . in all these studies , the use of a restrictive approach was associated with a decreased number of transfusions and , in most of them , with a decrease in the number of patients exposed to rbc transfusion . using a more liberal approach to achieve a higher hemoglobin concentration in an attempt to increase oxygen delivery and thus tissue oxygenation in stable critically ill patients does not appear to be associated with a significant clinical benefit . several authors have suggested that the rbc storage process could affect the ability of rbcs to transport and deliver oxygen , this phenomenon being responsible for the lack of apparent improvement in tissue oxygenation after transfusion . human studies on the effects of stored rbcs are scarce and controversial . in nine healthy volunteers undergoing acute isovolemic hemodilution , there were no differences in the ability of transfused fresh ( stored < 5 hours ) or stored ( > 3 weeks ) rbcs to reverse the neurocognitive deficit observed during acute anemia . in critically ill patients , the effect of rbc storage on gastric mucosal oxygenation remains controversial . in a randomized multicenter pilot trial , hbert and colleagues did not observe differences in mortality rates or life - threatening complications in patients transfused with fresh ( median age 4 days ) versus old ( median age 19 days ) rbcs . in the study of lacroix and colleagues , the average length of storage was about 16.0 10 days in both strategy groups . the effect of rbc storage time on primary outcome was not evaluated . for stable critically ill patients with a hemoglobin concentration ranging from 6 or 7 to 10 g / dl , there is increased evidence that a restrictive transfusion approach based on a predefined hemoglobin concentration does not influence outcome . the decision to transfuse such patients would , therefore , depend primarily on clinical judgment , taking into account the ability of the patient to increase cardiac output and oxygen extraction , and the level of tissue oxygen demand . it remains to be demonstrated that , in high risk patients , a symptomatic transfusion strategy is as effective , or possibly superior , to a hemoglobin - based transfusion strategy . this is the aim of the ongoing ' focus ' study comparing these two strategies in patients 50 years of age or older who undergo surgical repair of a hip fracture and who have clinical evidence for cardiovascular disease or cardiovascular risk factors . although the study of lacroix and colleagues adds an important piece to the ' puzzle ' , it still remains incomplete .

objectivesthis study determined the difference in the location of the caries dye staining endpoint of 1% acid red dye in propylene glycol versus that of 1% acid red dye in polypropylene glycol.methodsfreshly extracted permanent molar crowns with primary occlusal carious lesions were chisel - split axially to expose the lesion in cross - section on both halves . one half was stained with propylene glycol - based dye and the other with polypropylene glycol - based dye . for the control group , both halves were stained with propylene glycol - based dye . the dye staining front was marked on digital images of the stained split surfaces , and the images were aligned using reference notches . the distance between the marked staining front lines was measured in five locations , and the measurement protocol was repeated . weighted averages and a 95% confidence interval for the distance between marked staining front lines were calculated for the control and experimental groups.resultsthe weighted average distance for the experimental group ( 0.298 mm , 95% confidence interval 0.240 mm 0.357 mm ) was about four times that of the control group ( 0.070 mm , 95% confidence interval 0.051 mm 0.089 mm ) . generally , the marked staining line for the polypropylene glycol - based dye specimens was located shallow ( occlusal ) to the propylene glycol - based staining line ( range 0.12 mm to 0.66 mm).conclusionsthe staining endpoint of 1% acid red dye in polypropylene glycol is shallower than that of 1% acid red dye in propylene glycol . the method is useful for comparing staining endpoints of caries dye formulations . ( eur j dent 2008;2:2936 )

the removal of infected carious dentin is an exacting procedure routinely accomplished during tooth preparation , prior to placing a restoration . although explorer - verified hardness equal to that of normal dentin is traditionally considered the ideal endpoint of excavation , caries indicator dyes have been developed to provide a more objective and easily recognized visual differentiation for this endpoint.14 these dyes also distinguish between the outer layer of very soft highly infected carious dentin and the deeper layer of somewhat softened uninfected or at most lightly infected dentin.5,6 caries detector ( kuraray america , ny , ny ) and caries finder red ( danville materials , san ramon , ca ) are two representative products , consisting of 1% acid red 52 dye ( sulforhodamine b ) in propylene glycol.7 a recent approach to minimizing unwanted staining by caries indicator dyes has been to change the solvent for the dye compound . the rationale is based upon two studies that showed diminished diffusion into porous tissues of dyes dissolved in higher molecular weight solvents.810 instead of propylene glycol ( mw=76 ) ( pg ) or a mixture of pg and water as used in many currently marketed caries dye products , new formulations using larger molecules such as polypropylene glycol ( mw=300 , for example ) ( ppg ) have been proposed . in 2005 , a us patent application was filed for this type of caries detecting solution.11 this application discloses a range of inventive examples including solutions containing various mixtures of ppg with a molar weight of 300 , polyethylene glycol with molar weights of 400 and 4000 , pg , glycerin and water . two new caries indicator dye products are now available in japan ( caries check red and caries check blue , nishika co. , yamanashi , jp ) consisting of 1% acid red 52 or brilliant blue dye in 300 mw ppg . a few studies do indicate , at least indirectly , that ppg - based dye solutions may provide a more conservative excavation endpoint . when the final excavated surfaces determined by pg - based dye solution or ppg - based dye solution were compared using micro - vickers hardness ( mvh ) , it was found that the ppg dye - determined group was significantly softer than the pg dye - determined group ( mean mvh 32.7 vs. 44.7).12 since hardness generally increases with increasing depth in dentin carious lesions this result would suggest a shallower , more conservative endpoint for the ppg dye - determined specimens.3 because both groups were found to be free of bacteria histologically , it was concluded that the ppg dye - determined endpoint would be preferable . however , since hardness generally decreases with increasing depth in normal dentin , the clinical interpretation of hardness differences is somewhat obscured . after this initial report , studies incorporating laser fluorescence comparisons pointed toward a similar conclusion , at least for permanent teeth . the rationale for using laser fluorescence values ( dd ) ( diagnodent , kavo , usa ) to assess differences in excavation endpoints was based upon a study of the spectral autofluorescence characteristics of carious lesions , as well as general guidelines for interpretation of dd values when detecting carious lesions in occlusal fissures.1315 in extracted carious teeth , it was shown that the mean mvh was significantly lower and dd values significantly higher in the ppg dye - determined group versus the pg dye - determined group.16 in another study , a similar result was found for the group mean dd value in permanent teeth but not in primary teeth.17 another study using extracted teeth containing acute or chronic carious lesions also found that the mean dd value was higher for the ppg dye - determined group versus the pg dye - determined group.13 in a clinical study , this same result regarding dd values was confirmed for permanent teeth but not for primary teeth.8 one of these studies also found that all specimens first excavated to an endpoint indicated with the ppg - based dye could subsequently be re - stained with the pg - based dye , once again suggesting that ppg - based dye solutions may indicate a more conservative endpoint for excavation.13 more direct confirmation of this apparently more conservative endpoint indicated by ppg - based dyes is necessary for several reasons . first , although these initial studies do demonstrate a statistically significant difference in mean mvh and dd value between sample groups , it is not clear whether a difference exists in all carious lesions or just certain ones . in addition , although microhardness does generally increase with increasing depth in a dentin carious lesion , the relationship between microhardness and depth is neither linear nor constant from tooth to tooth.18 similarly , although dd values do generally decrease with increasing depth in a dentin carious lesion , this inverse relationship is not linear and certainly not constant from tooth to tooth.19 this is true both at the unexcavated lesion surface and across the pg dye - guided excavation front.19 in light of these variables , and to assess clinical implications for cavity preparation , it is necessary to investigate the differences between the actual physical locations of surfaces created by ppg dye - guided excavation and those created by pg dye - guided excavation . the purpose of this study is to determine the difference in the location of the caries dye staining endpoint indicated by 1% acid red 52 dye in pg versus that indicated by 1% acid red 52 dye in ppg , in freshly extracted human permanent posterior teeth containing primary dentin carious lesions . dye solutions were made by completely dissolving 0.100 g acid red 52 ( sulforhodamine b , # s-9012 , sigma chemical co. , st . louis , mo ) in 10.00 ml propylene glycol ( sigma # p-1009 ) or in 10.00 ml polypropylene glycol , ( triol type , mw=300 ) ( # 160 - 17605 , wako chemicals usa , richmond , va ) at room temperature . solutions were stirred by hand for ten minutes and vortex - mixed for two minutes twenty - four hours later . freshly extracted , unrestored human permanent posterior teeth with primary occlusal carious lesions were collected under an irb - exempt protocol . teeth were stored in sterile distilled water at 4c for up to two weeks prior to processing . the outer surface of each specimen was cleaned with a soft - bristle toothbrush and sterile distilled water , sectioned horizontally at the dentino - enamel junction , the root portion discarded and the pulp tissue removed from the crown portion . two opposing axial surfaces were ground flat , parallel to each other and perpendicular to the previous horizontal section plane using an 8 240 grit carborundum disc mounted in a water - lubricated surface polisher ( model 900 , electron microscopy sciences , hatfield , pa ) . in preparation for splitting , a continuous fine groove was cut about 1 mm deep in the center of each ground axial surface running occlusogingivally and connecting across the occlusal surface approximately bisecting the external surface of the carious lesion . the groove was made with a 1 thin diamond disc mounted in a lowspeed handpiece . to provide identical reference marks on both split tooth halves , two to six orientation notches about 2 mm deep were made with a # 57 carbide bur across and perpendicular to the fine groove at various locations , but not in the region of the carious lesion . the crown specimens were fractured along the groove using a 3/8 steel chisel and mallet yielding two segments , each containing a split surface showing the carious lesion in cross - section . one of the two surfaces from each crown specimen , chosen at random , was stained with the ppg - based dye and the other with the pg - based dye . two drops of dye were applied for ten seconds , rinsed under running tap water for ten seconds , the surface blotted damp - dry with a cotton gauze square and then observed under 2.6x loupe magnification . specimen pairs were discarded if they had not fractured along the fine groove or were missing tooth structure , if no lesion was observed in dentin or if the lesion extended to the pulp chamber , if the lesion was too dark to permit differentiation of the red dye staining , if the lesion was less than 1 mm into dentin , or if a previously unobserved restoration was detected . of the 41 specimens originally collected , 18 were accepted for further analysis . as controls , three of the 18 fractured specimen pairs were stained on both surfaces with 1% sulforhodamine b in pg . color digital images were then made of each fracture surface . using a digital microscope and camera ( optem zoom-100 , qioptiq , rochester , ny ) ( dp-11 , olympus america , melville , ny ) at 10x magnification and with ring light illumination ( # eke , schott - fostec , auburn , ny ) , image files were created with the following settings : white balance 3000k , resolution set at hq 17121368 pixels , iso 100 , ring light brightness 70 , and 1/15 sec exposure . images were saved as.jpg files . to standardize the orientation of the fracture surface relative to the camera , both a machinist square was fastened to the stand base to fix the position of a removable aluminum block on which specimens were mounted using a thin layer of boxing wax . for linear calibration , a millimeter grid was fixed to the block . image pairs were analyzed using a digital image analysis software program ( image pro discovery 4.5 , media cybernetics , silver spring , md ) . for each specimen , one of the two image pairs was opened , flipped left - to - right , and magnified 3x . using a graphic tablet and digital pen ( intuos 3 , wacom , vancouver , wa ) the most pulpal extent of the red dye - staining the other image of the pair was then opened , magnified 3x , and the pulpal extent of the dye - staining marked with a one pixel - wide blue line . this was accomplished by selecting a rectangular region of interest on one image that included both the blue or yellow line as well as the millimeter reference grid , edge of the aluminum mounting block and reference notches in the specimen surface . the selected area was copied and pasted into the other image using a 50% translucency setting for the paste operation , providing a means to visually align the two image pairs on the reference notch edges and aluminum mounting block edge , while permitting both the blue and yellow marked lines to be clearly seen . the distance between the yellow and blue lines was then measured using the software s linear measurement tool at 2x magnification after calibration based on the millimeter grid image . distance was measured from line to line ( l - l distance ) at five locations beginning in the center of the lesion , with two additional measurements on either side , spaced 0.5 mm apart , perpendicular to the most occlusally located line and recorded in millimeters to two decimals . this measurement protocol was repeated once for all 15 experimental samples and for two of the three control samples , providing a total of 150 experimental ( ten per sample ) and 50 ( five or ten per sample ) control l - l distance measurements . for each of the control ( 6 ) and experimental ( 15 ) specimen image pairs , mean l - l distance values were calculated . using these means , a weighted average was calculated for the combined control group and for the combined experimental group . the weighted averages were based on adjustment of the six or fifteen mean l - l distance values from each specimen group by the variance of the individual l - l distance values within that group . a 95% confidence interval was calculated for the weighted averages . to evaluate the test method , a 3-factor anova ( specimen , measurement , replication ) on rank - transformed data was carried out on the control and experimental data from the individual specimens ( control 6 , experimental 15 ) , using repeated measurements of each test pair ( 5 ) , and replication of the measurement protocol ( 2 ) . prior to the anova , the data in both the control and experimental groups were analyzed for normality using the shapiro - wilk test . all statistical analysis used sas v9.1 software ( sas institute , cary , nc ) . for 12 of the 15 experimental specimen image pairs , the line corresponding to ppg dye staining was located more occlusally ( shallower ) throughout its length than the line corresponding to pg dye staining . for two image pairs , the ppg dye staining line was predominately located more occlusally , but there were areas where the two lines were coincident or where the pg dye staining line was shallower . for these two specimen image pairs , this was true for both iterations of the image analysis protocol . in the specimen # 4 image pair , the pg dye staining line was shallower for 12% ( first iteration ) and 51% ( second iteration ) of the line length , and coincident for 11% ( first iteration only ) . in the specimen # 8 image pair , the pg dye staining line was shallower for 14% and 19% of the line length and coincident for 5% and 13% , respectively . for one specimen image pair ( # 1 ) , the dye staining lines were coincident for 2% of the line distance in one iteration only , and elsewhere the ppg dye staining line was shallower . mean l - l distances were small , ranging from 0.0377 mm to 0.0952 mm . mean l - l distances were larger than for the controls , ranging from 0.153 mm to 0.506 mm , while individual distances ranged from ( ) 0.12 mm to ( + ) 0.66 mm . table 3 presents the data for the weighted averages of the means for the control and experimental specimen image pairs . for the control specimen image pairs , the weighted average l - l distance was 0.70 mm , with a 95% confidence interval of 0.51 mm to 0.89 mm , versus a 0.298 mm l - l distance and 95% confidence interval of 0.2140 mm to 0.357 mm for the experimental group . the weighted average l - l distance for the experimental group was about four times that of the control group , with no overlap and 0.151 mm between the two confidence intervals . the shapiro - wilk test for normality indicated that the control data was normally distributed ( p=.1226 ) but that the experimental data was not normally distributed ( p=.0003 ) . therefore , anova on ranks was used to detect effects by groups . at a significance level of p=.05 , the anova on ranks detected a significant effect by specimen and measurement , but not by replication in the control data . table 4 summarizes the anova on ranks results by effects for control and experimental groups . the 0.298 mm weighted average l - l distance for all experimental specimens is clinically relevant since it is possible to remove dentin in increments of this magnitude , potentially resulting in more conservative cavity preparations and even preventing unnecessary pulp exposures in some instances . this advantage is somewhat limited , however , because 0.3 mm represents perhaps one or two careful applications of a hand excavator or round bur , and because the l - l distance ranged considerably : from 0.12 mm to 0.66 mm within the specimens . with an aggressive excavation technique exceeding 0.3 mm or even less per increment , any advantage from a more conservative ppg - based caries dye endpoint would often be negated . however , in some cases , careful excavation using this ppg - based dye could actually prevent exposure in deep lesions approaching the pulp . because carious lesions differ in type and amount of bacterial load , nutrient availability , and morphology and because dentin itself varies according location , age and reaction pattern , considerable variation would be expected in l - l distances among other specimen sets such as primary teeth , more chronic lesions , very active lesions , root lesions , and lesions associated with restorations . the results of this study should be interpreted with caution , especially for clinical application . since no difference was found in dd values for excavation surfaces created using pg - based versus ppg - based dyes in primary teeth in two previous studies , it may be that no l - l distance difference exists in primary teeth . for this reason , the present study needs to be repeated for primary teeth.8,17 other solvents or solvent mixtures as well as dyes other than sulforhodamine b may produce different results , and would also need to be tested . the fractured dentin surface used in the present study does not include a smear layer as would sometimes be produced clinically by burs or hand excavators , and may , therefore , indicate a more conservative endpoint since a smear layer might stain with dye , having originated from elsewhere in the preparation . therefore , the present study may represent a more idealized staining result compared to some clinical situations . the differences in the weighted averages between the control and experimental groups as well as the results on the anova on ranks analysis indicate that the present method is useful for differentiating staining endpoints between caries dyes . the method was able to detect a relatively small l - l distance , approximately four times the average error seen in the control group which itself was very small . in both groups , a specimen effect was detected by anova as would be expected for clinical specimens varying in lesion location , activity , size , etc . effect was detected in the control group and almost in the experimental group ( p=0.0510 ) , also expected due to lesser variations in measurement orientation versus dentin tubule orientation , for example . effect was not detected in either group , indicating an acceptable degree of consistency between runs . for future studies , errors might be reduced by more precise machining of the splitting groove and orientation notches , and by color - corrected thresholding of the stained specimen surface . in the present study , any light pink staining encountered on the dye - stained fracture surface was treated as non - stained , and therefore the dye - stain demarcation line was drawn shallow to the light pink staining , at the border of red and light pink staining . in previous studies using dd or mvh to detect endpoint differences in pg - based versus ppg - based caries dyes , the light pink staining in the pg group was sometimes kept in place and sometimes removed prior to surface testing.8,13,16,17 in the present study , had the dye - stain demarcation line been drawn at the border of non - stained and light pink - stained dentin , l - l distances would have been greater in some instances . further study is indicated to determine the tendency of ppg - based dyes to stain dentin light pink , and then to characterize those areas according to hardness , bacterial load , etc . there are no long - term clinical trials showing improved clinical outcomes for any dye - based excavation endpoints versus conventionally - determined endpoints , let alone for any one type of dye solution . in fact , clinical studies comparing outcomes for excavation methods in general are quite limited . in one clinical study of restorations in primary teeth , no differences were found at one year regarding marginal adaptation and secondary caries in the chemo - mechanical excavation group compared to the conventional hand excavation group.20 in another study of restorations in primary teeth followed for six months , no difference was detected in the survival rate of restorations in the chemo - mechanical excavation group compared to the conventional bur excavation group.21 factors such as restoration longevity , margin integrity , tooth structure integrity , pulpal health , and secondary caries are all relevant considerations . in this regard , ppg - based dye excavation should also be compared to other excavation methods such as chemo - mechanical , polymer bur , and fluorescence - aided excavation , in vitro and clinically.2224 until such studies are completed , the optimal endpoint for excavation of carious dentin will remain a matter of debate , based primarily on indirect evidence . under the experimental conditions and compared to pg - based sulforhodamine b caries dye solution , ppg - based dye solution provides a significantly shallower excavation endpoint by 0.298 mm ( 95% confidence interval 0.240 mm

heterogeneous diffusion dynamics of molecules play an important role in many cellular signaling events , such as of lipids in plasma membrane bioactivity . however , these dynamics can often only be visualized by single - molecule and super - resolution optical microscopy techniques . using fluorescence lifetime correlation spectroscopy ( flcs , an extension of fluorescence correlation spectroscopy , fcs ) on a super - resolution stimulated emission depletion ( sted ) microscope , we here extend previous observations of nanoscale lipid dynamics in the plasma membrane of living mammalian cells . sted - flcs allows an improved determination of spatiotemporal heterogeneity in molecular diffusion and interaction dynamics via a novel gated detection scheme , as demonstrated by a comparison between sted - flcs and previous conventional sted - fcs recordings on fluorescent phosphoglycerolipid and sphingolipid analogues in the plasma membrane of live mammalian cells . the sted - flcs data indicate that biophysical and biochemical parameters such as the affinity for molecular complexes strongly change over space and time within a few seconds . drug treatment for cholesterol depletion or actin cytoskeleton depolymerization not only results in the already previously observed decreased affinity for molecular interactions but also in a slight reduction of the spatiotemporal heterogeneity . sted - flcs specifically demonstrates a significant improvement over previous gated sted - fcs experiments and with its improved spatial and temporal resolution is a novel tool for investigating how heterogeneities of the cellular plasma membrane may regulate biofunctionality .

lipolysis is defined as the catabolism of triacylglycerols stored in cellular lipid droplets . recent discoveries of essential lipolytic enzymes and characterization of numerous regulatory proteins and mechanisms have fundamentally changed our perception of lipolysis and its impact on cellular metabolism . new findings that lipolytic products and intermediates participate in cellular signaling processes and that lipolytic signaling is particularly important in many nonadipose tissues unveil a previously underappreciated aspect of lipolysis , which may be relevant for human disease .

lipolysis describes the hydrolysis of triacylglycerols ( tgs ) , commonly referred to as fat . in the mid-19 century the great french physiologist claude bernard ( bernard , 1856 ) noted that the pancreatic juice of mammals was able to efficiently degrade fat in the form of butter and oil . his observation led to the partial characterization of a pancreatic fat - splitting ferment by balser ( balser , 1882 ) , langerhans ( langerhans , 1890 ) , and flexner ( flexner , 1897 ) . the importance of lipolysis to general metabolism became apparent when whitehead ( whitehead , 1909 ) discovered that fat ( tgs ) could not enter cells in its unhydrolyzed form . the absolute requirement for tg hydrolysis for the cellular uptake or release of fatty acids ( fas ) and glycerol defines three processes in vertebrate physiology where lipolysis is essential : gastrointestinal lipolysis mediates the catabolism of dietary fat ; vascular lipolysis is responsible for the hydrolysis of lipoprotein - associated tgs in the blood ; and intracellular lipolysis catalyzes the breakdown of tgs stored in intracellular lipid droplets ( lds ) for subsequent export of fas ( from adipose tissue ) or their metabolism ( in nonadipose tissues ) . although fundamental aspects of lipolysis were understood early on , it took more than a century to identify , isolate , and characterize the main fat - splitting ferments , which have been called lipases since 1900 . the chief lipolytic enzymes of the gastrointestinal tract are lingual lipase , gastric lipase , pancreatic lipase , and pancreatic lipase - related proteins 1 , 2 , and 3 . vascular tg hydrolysis depends on lipoprotein lipase ( lpl ) and hepatic tg lipase . intracellular lipolysis of tgs involves neutral ( ph - optimum around ph 7 ) and acid lipases present in lysosomes ( ph optimum between ph 45 ) . well - characterized neutral tg hydrolases include adipose triglyceride lipase ( atgl ) and hormone - sensitive lipase ( hsl ) , whereas lysosomal acid lipase ( lal ) is the most important lipase in lysosomes . besides an active serine site , these enzymes share no obvious structural homologies and are unrelated to the pancreatic lipase family . lipolysis is the catabolic branch of the fa cycle that provides fas in times of metabolic need and removes them when they are present in excess . fas are essential as substrates for energy production and the synthesis of most lipids , including membrane lipids and lipids involved in cellular signaling . accordingly , all uni- and multicellular organisms are able to synthesize fas de novo ( endogenous fas ) from carbohydrate and/or protein metabolites . despite their fundamental physiological importance , an oversupply of fas is highly detrimental . increased concentrations of nonesterified fas disrupt the integrity of biological membranes , alter cellular acid - base homeostasis , and elicit the generation of harmful bioactive lipids . these effects , in turn , impair membrane function and induce endoplasmic reticulum ( er ) stress , mitochondrial dysfunction , inflammation , and cell death . collectively , these deleterious effects are subsumed under the term lipotoxicity ( unger et al . , 2010 ) . as a countermeasure , essentially all cells are able to detoxify nonesterified fas by esterification with glycerol to yield inert tgs . additionally , higher organisms store fas in a specialized organ ( i.e. , adipose tissue ) , which supplies fas to other high - demand tissues , such as liver and muscle ( exogenous fas ) . the carefully regulated balance of fa esterification and tg hydrolysis creates an efficient buffer system , allowing sufficient fa flux without nonphysiological increases in cellular nonesterified fa concentrations moreover , fa cycling creates metabolic intermediates that can be utilized in anabolic processes or as extra- or intracellular signaling molecules . we summarize recent advances in understanding the enzymatic mechanisms of the lipolytic process and the ( patho)physiological impact of lipolysis on energy homeostasis and cellular signaling . neutral hydrolysis of tgs to fas and glycerol requires three consecutive steps that involve at least three different enzymes : atgl catalyzes the initial step of lipolysis , converting tgs to diacylglycerols ( dgs ) ; hsl is mainly responsible for the hydrolysis of dgs to monoacylglycerols ( mgs ) and mg lipase ( mgl ) hydrolyzes mgs . in adipose tissue , atgl and hsl are responsible for more than 90% of tg hydrolysis ( schweiger et al . , 2006 ) . although most nonadipose tissues also express atgl and hsl , expression levels are low in some tissues , raising the question of whether other lipases are additionally required for efficient lipolysis . , 2004 ; villena et al . , 2004 ; zimmermann et al . , 2004 ) , belongs to the family patatin domain - containing proteins including nine human and eight murine members . the patatin domain was originally discovered in lipid hydrolases of the potato and other plants and named after the most abundant protein of the potato tuber , patatin . because some members of the family act as phospholipases , the proteins were officially named patatin - like phospholipase domain - containing protein a1 to a9 ( pnpla19 ) ( wilson et al . , 2006 ) . unfortunately , this name is misleading because it implies that all members of this family are phospholipases . however , several pnpla proteins have only minor or no phospholipase activity . a more appropriate designation for the family orthologous enzymes are found in essentially all eukaryotic species , including vertebrates , invertebrates , plants , and fungi . , the active site of the enzyme contains an unusual catalytic dyad ( s47 and d166 ) within the patatin domain ( rydel et al . , 2003 ) . this domain comprises 180 aa and is embedded within a 250 aa sandwich structure at the protein 's nh2-terminal half . the cooh - terminal half has a predominantly regulatory function and contains a predicted hydrophobic region for ld binding ( duncan et al . , 2010 ; schweiger et al . , loss of this region increases the specific in vitro activity of atgl against artificial tg substrates but blunts the intracellular activity due to the inability of the truncated enzyme to bind to cellular lds . regulation of atgl expression and enzyme activity is complex ( lass et al . , 2011 ) . atgl mrna expression is elevated by peroxisome proliferator - activated receptor ( ppar ) agonists , glucocorticoids , and fasting , whereas insulin and food intake decrease expression . more recently , it was shown that mtor complex 1-dependent signaling reduces atgl mrna levels ( chakrabarti et al . , 2010 ) . conversely , activation of foxo1 by sirt1-mediated deacetylation activates lipolysis by increasing atgl mrna levels . sirt1 silencing has the opposite effect ( chakrabarti et al . , 2011 ) . the abundance of atgl ( and hsl ) mrna does not always correlate with cellular lipase activity . for example , isoproterenol and tumor necrosis factor- reduce atgl ( and hsl ) mrna levels in adipocytes ( kralisch et al . , 2005 ) but , conversely , stimulate lipase activities and fa and glycerol release . the discrepancy between enzyme mrna levels and activities is explained by the extensive posttranslational regulation of atgl and hsl ( discussed below ) . at least two serine residues in atgl can be phosphorylated ( s406 and s430 in the murine enzyme ) ( bartz et al . , 2007 ) . in contrast to hsl ( see below ) , atgl modification is not protein kinase a ( pka)-dependent ( zimmermann et al . , 2004 ) . sul recently showed that amp - activated kinase ( ampk ) phosphorylates s406 , leading to increased hydrolytic activity of murine atgl ( ahmadian et al . , 2011 ) . the role of ampk in the regulation of lipolysis has been controversial , with data showing that ampk induces ( gaidhu et al . , 2009 ; yin et al . , 2003 ) , inhibits ( daval et al . , 2005 ; gauthier et al . , 2008 ) , or has no effect ( chakrabarti et al . , 2011 ) on lipolysis . interestingly , in the nematode caenorhabiditis ( c. ) elegans , ampk - mediated phosphorylation of atgl-1 ( the worm ortholog of mammalian atgl ) at different phosphorylation sites inhibits tg hydrolysis ( narbonne and roy , 2009 ) , delays the consumption of tg stores , and prolongs life span during the stress - induced dauer larval stage . ( cgi-58 ) , for full hydrolase activity ( lass et al . , 2006 ) . cgi-58 was originally discovered in a screen comparing the proteomes of humans and c. elegans . the gene is now officially named / hydrolase domain - containing protein-5 ( abhd5 ) , owing to the presence of an / hydrolase domain commonly found in esterases , thioesterases , and lipases . however , cgi-58 is unlikely to exhibit hydrolase activity due to the fact that asparagine-153 ( n153 ) in cgi-58 replaces a nucleophilic serine residue that is required in the active site of enzymatically functional members of the esterase / thioesterase / lipase family . interestingly , substitution of n153 by serine does not convert cgi-58 into a lipid hydrolase for tgs , dgs , mgs , or short fa - glycerol esters ( a. lass et al . , unpublished ) . in the epidermis of the skin , cgi-58 may also stimulate another , currently unknown , tg hydrolase distinct from atgl ( radner et al . , 2010 ) . importantly , two laboratories showed that cgi-58 , at least in vitro , exhibits enzymatic activity as an acylcoa - dependent acylglycerol-3-phosphate acyltransferase ( agpat ) ( ghosh et al . it may affect phosphatidic acid or lysophosphatidic acid signaling ( brown et al . , 2010 ) . recently , ( yang et al . , 2010b ) discovered a specific peptide inhibitor for atgl . the protein was originally identified in blood mononuclear cells to act at the g0 to g1 transition of the cell cycle . consistent with this function , it was named g0g1 switch protein 2 ( g0s2 ) . human and murine g0s2 have a predicted primary structure of 103 aa . the protein is found in many tissues , with highest concentrations in adipose tissue and liver . consistent with lipase activities , g0s2 expression is very low in adipose tissue during fasting but increases after feeding ( yang et al . , 2010b ) . conversely , fasting or ppar-agonists increase hepatic g0s2 expression ( zandbergen et al . , 2005 ) . the protein localizes to multiple cellular compartments , including lds , cytoplasm , er , and mitochondria . the different localizations may reflect multiple functions for g0s2 in regulating lipolysis , the cell cycle , and , possibly , apoptosis via its ability to interact with the mitochondrial antiapoptotic factor bcl2 ( welch et al . , 2009 ) . in vitro , ld binding and inhibition of atgl depend on a physical interaction between the nh2-terminal region of g0s2 ( involving aa 2742 ) and the patatin domain of atgl ( lu et al . , 2010 ) . elucidation of whether g0s2 also regulates tissue - specific lipolysis in vivo will require the characterization of g0s2 transgenic and knockout mouse models . ld - associated proteins participate in the cgi-58-mediated regulation of atgl ( figure 1 , left ) . in hormonally nonstimulated white and brown adipocytes , perilipin-1 interacts with cgi-58 , preventing its binding to and , thus , induction of atgl . upon -adrenergic stimulation , protein kinase a ( pka ) phosphorylates perilipin-1 at multiple sites , including the critical serine-517 residue , causing the release of cgi-58 . thus , -adrenergic stimulation of pka induces atgl activity by phosphorylation of perilipin-1 and not by direct enzyme phosphorylation . consistent with this model , frameshift mutants of perilipin-1 in humans ( l404fs and v398fs ) fail to bind cgi-58 , leading to unrestrained lipolysis , partial lipodystrophy , hypertriglyceridemia , and insulin resistance ( gandotra et al . , 2011 ) . atgl - mediated tg hydrolysis in nonadipose tissues with high fa oxidation rates , such as muscle and liver , follows another mechanism . in these tissues , perilipin-1 is replaced by perilipin-5 ( figure 1 , right ) . during fasting , perilipin-5 recruits both atgl and cgi-58 to lds by direct binding of the enzyme and its coactivator . formation of the ternary complex involves the cooh - terminal region of perilipin-5 ( aa 200463 ) ( granneman et al . , 2011 ) . recent data suggest that it is involved in the interaction of lds with mitochondria and inhibits atgl - mediated tg hydrolysis ( wang et al . , 2011 ) . whether perilipins-2 , -3 , and -4 also interact with either atgl or cgi-58 is disputed . overexpression of perilipin-2 in hepatocyte cell lines inhibits atgl activity by restricting its physical access to lds . direct protein - protein interaction is probably not required ( bell et al . , 2008 ; listenberger et al . , 2007 ) . recently , several groups reported that pigment epithelium - derived factor ( pedf ) induces tg hydrolysis in adipose tissue , muscle , and liver , via atgl ( borg et al . , 2011 ; pedf is a widely expressed 50 kd protein of the noninhibitory serpin family of serine protease inhibitors ( filleur et al . , 2009 ) . it exhibits a large spectrum of bioactivities , including antiangiogenic , antitumorigenic , neuroprotective , antioxidative , and antiinflammatory effects . pedf binds to atgl and activates its enzymatic activity ( borg et al . , 2011 ; notari et al . , 2006 although the mechanism remains to be clarified , atgl activation by pedf may be involved in the pathogenesis of insulin resistance and the development of hepatosteatosis . another important aspect of atgl regulation was identified by genetic analyses of ld formation in drosophila ( d. ) melanogaster l2 cells ( beller et al . , 2008 ; guo et al . , compelling results showed that atgl delivery to lds requires functional vesicular transport . in the absence of essential protein components of the transport machinery , such as adp - ribosylation factor 1 ( arf1 ) , small gtp - binding protein 1 ( sar1 ) , the guanine - nucleotide exchange factor golgi - brefeldin a resistance factor ( gbf1 ) , or deficiency of the coatamer protein coat - complex i and ii , atgl translocation to lds is blocked and the enzyme remains associated with the er ( soni et al . , 2009 ) . the process requires physical binding of atgl to gbf1 ( ellong et al . , 2011 ) . in the early 1960s , it was noted that a lipolytic activity present in adipose tissue was induced by hormonal stimulation . a landmark paper published by d. steinberg 's group ( vaughan et al . , 1964 ) although this classic work originally noted that hsl is a much better dg hydrolase than tg hydrolase , standard textbook knowledge perpetuated the conclusion that hsl was rate - limiting for the catabolism of fat stores in adipose and many nonadipose tissues . this view required revision when hsl - deficient mice efficiently hydrolyzed tgs ( osuga et al . , 2000 ) . hsl - deficient mice showed no signs of tg accumulation in either adipose or nonadipose tissues ; instead , they accumualted large amounts of dgs in many tissues , suggesting that in vivo the enzyme was more important as a dg- than a tg - hydrolase ( haemmerle et al . , 2002 ) . although originally somewhat controversial ( rydn et al . , 2007 ) , it is now accepted that atgl is responsible for the initial step of lipolysis in human adipocytes , and that hsl is rate - limiting for the catabolism of dgs ( bezaire et al . , 2009 ) . in addition to dgs , hsl also hydrolyzes ester bonds of many other lipids ( e.g. , tgs , mgs , cholesteryl esters , and retinyl esters ) and short - chain carbonic acid esters ( fredrikson et al . , 1986 ) . highest mrna and protein concentrations are found in white adipose tissue ( wat ) and brown adipose tissue ( bat ) ; low expression is detected in many other tissues , including muscle , testis , steroidogenic tissues , and pancreatic islets ( holm et al . , 2000 ) . alternative exon usage leads to tissue - specific differences in mrna and protein size ( holst et al . , in adipose tissue , the hsl protein comprises 768 aa . unlike atgl , with orthologous enzymes found across all eukarya , hsl is less ubiquitous phylogenetically . for example , no hsl ortholog is known in birds , c. elegans , d. melanogaster , and saccharomyces ( s. ) cerevisiae . interestingly , the closest structural relatives to hsl are found in prokaryotes ( e.g. , lipase 2 in moraxella ta144 ) ( langin et al . , 1993 ) . functional studies have delineated in hsl an nh2-terminal lipid - binding region , the / hydrolase fold domain including the catalytic triad , and the regulatory module containing all known phosphorylation sites important for regulation of enzyme activity ( holm et al . , 2000 ) . since atgl and hsl hydrolyze tgs in a coordinated manner , it is not unexpected that they share many regulatory similarities . in adipose tissue , hsl enzyme activity is strongly induced by -adrenergic stimulation , whereas insulin has a strong inhibitory effect . while -adrenergic stimulation regulates atgl primarily via recruitment of the coactivator cgi-58 ( see above ) , hsl is a major target for pka - catalyzed phosphorylation ( strlfors and belfrage , 1983 ) . other kinases , including ampk , extracellular signal - regulated kinase , glycogen synthase kinase-4 , and ca / calmodulin - dependent kinase , also phosphorylate hsl to modulate its enzyme activity ( lass et al . , the enzyme has at least five potential phosphorylation sites , of which s660 and s663 appear to be particularly important for hydrolytic activity ( anthonsen et al . , 1998 ) . enzyme phosphorylation affects enzyme activity moderately ( an approximate 2-fold induction ) . for full activation , hsl must gain access to lds , which , in adipose tissue , is mediated by perilipin-1 . simultaneously with hsl , pka also phosphorylates perilipin-1 on six consensus serine residues . as a result , hsl binds to the nh2-terminal region of perilipin-1 , thereby gaining access to lds ( miyoshi et al . , 2007 ; shen et al . , 2009 ; wang et al . , 2009 ) . together , hsl - phosphorylation and enzyme translocation to lds coupled with atgl activation by cgi-58 result in a more than 100-fold increase in tg hydrolysis in adipocytes ( figure 1 ) . for example , receptor - interacting protein 140 ( rip-140 ) was shown to induce lipolysis by binding to perilipin-1 , increasing hsl translocation to lds , and activating atgl via cgi-58 dissociation from perilipin-1 ( ho et al . , 2011 ) . in nonadipose tissues , such as skeletal muscle , hsl is activated by phosphorylation in response to adrenaline and muscle contraction ( watt et al . , 2006 ) . these tissues lack perilipin-1 , and it remains to be determined which alternative mechanisms regulate hsl access to lds . insulin - mediated deactivation of lipolysis is associated with transcriptional downregulation of atgl and hsl expression ( kershaw et al . , 2006 ; kralisch et al . , additionally , insulin signaling results in phosphorylation and activation of various phosphodiesterase ( pde ) isoforms by pkb / akt ( enoksson et al . , 1998 ) , pde - catalyzed hydrolysis of camp , and inhibition of pka . these actions halt lipolysis by preventing phosphorylation of both hsl and perilipin-1 , activation and translocation of hsl , and activation of atgl by cgi-58 . in addition to its peripheral action , insulin also acts centrally via the sympathetic nervous system to inhibit lipolysis in wat . elegant studies by scherer and coworkers ( scherer et al . , 2011 ) showed that increased insulin levels in the brain inhibit hsl and perilipin phosphorylation , leading to reduced hsl and atgl activities . mgl is considered to be the rate - limiting enzyme for the breakdown of mgs derived from extracellular tg hydrolysis ( by lpl ) , intracellular tg hydrolysis ( by atgl and hsl ) , and intracellular phospholipid hydrolysis ( by phospholipase c and membrane - associated dg lipase and ) . the enzyme localizes to cell membranes , cytoplasma ( sakurada and noma , 1981 ) , and lds ( unpublished data ) . mgl received significant attention following the realization that glycerophospholipid - derived mg 2-arachidonylglycerol ( 2-ag ) is a major agonist for endocannabinoid signaling and is inactivated by the hydrolytic activity of mgl ( see mg - signaling , below ) . the importance of mgl for efficient degradation of mgs was recently confirmed in mutant mouse models ( chanda et al . , 2010 ; schlosburg et al . , 2010 ; taschler et al . , lack of mgl impairs lipolysis and is associated with increased mg levels in adipose and nonadipose tissues alike . mgl shares homology with esterases , lysophospholipases , and haloperoxidases , and contains a consensus gxsxg motif within a catalytic triad ( s122 , a239 , and h269 for mouse mgl ) that is typical of lipases and esterases . very recently , the crystal structure of mgl was solved ( bertrand et al . , 2010 ; the enzyme exhibits the classic fold of the / hydrolases , crystallizes as a dimer , and exhibits a wide , hydrophobic access to the catalytic site . an apolar helix - domain lid covers the active site and mediates the interaction of mgl with membrane structures and the recruitment of substrate . experiments with atgl - deficient mice and small - molecule hsl inhibitors revealed that atgl and hsl are responsible for more than 90% of the lipolytic activity in wat and cultured adipocytes ( schweiger et al . , 2006 ) . in nonadipose tissues , the contribution of other neutral lipases to the catabolism of stored tgs may be more prominent . for example , in the liver of fasted mice , atgl accounts for less than 50% of neutral tg hydrolase activity ( reid et al . , 2008 ) . this activity is physiologically important because atgl - deficient mice develop hepatosteatosis ( haemmerle et al . however , the pronounced remaining activity of hepatic tg hydrolase(s ) in atgl - deficient mice indicates that other lipases contribute to the highly dynamic turnover of tgs . this view is supported by the observation that mice lacking atgl in the liver have no apparent defect in vldl biogenesis ( wu et al . , 2011 ) , although assembly and secretion of hepatic vldl particles require substantial mobilization of hepatic tg stores . because hsl is also poorly expressed in hepatocytes several members of the carboxylesterase / lipase family and the pnpla family have been suggested as potential tg hydrolases . one of them , carboxyl esterase-3/triglyceride hydrolase-1 ( ces-3/tgh-1 , ortholog of human ces-1 ) , has gained major interest because the recent characterization of ces-3/tgh-1-deficient mice provided compelling evidence that the enzyme participates in the assembly and secretion of hepatic vldl ( wei et al . , 2010 ) . how this biological function conforms to the strict luminal localization of tgh-1 in the er remains to be elucidated . structural relatives of atgl within the pnpla family were also considered as potential tg hydrolases . for example , pnpla4 and -5 exhibit tg - hydrolase , dg transacylase , and retinylester hydrolase activity in vitro ( kienesberger et al . , the member with highest homology to atgl is adiponutrin ( pnpla3 ) , with over 50% aa identity within the patatin domain . adiponutrin was originally discovered as a nutritionally regulated adipose - specific transcript of unknown function ( baulande et al . , 2001 ) . interest in adiponutrin increased tremendously when h. hobbs and colleagues found a strong genetic association between a nonsynonymous aa change ( i148 m ) in adiponutrin and susceptibility to develop nonalcoholic fatty liver disease ( nafld ) ( romeo et al . , 2008 ) . several other groups confirmed and extended this important finding by showing robust associations of i148 m with alcoholic- and nonalcoholic liver disease , hepatic fibrosis , and liver cirrhosis ( krawczyk et al . , 2011 ; tian et al . , 2010 ; the close similarity to atgl as well as the presence of conserved structural motifs typical for lipases / esterases ( sandwich structure and the gxsxg motif within a catalytic dyad ) suggest a lipase function for adiponutrin . in accordance with this assumption , several groups reported that adiponutrin acts as a tg hydrolase and additionally exhibits dg transacylase activity ( he et al . , 2010 ; huang et al . , 2011 ; jenkins et al . , 2004 ; lake et al . , however , the expression profile generated in response to fasting / feeding and the induction of adiponutrin gene expression by srebp1a / c and chrebp argued against an in vivo role of adiponutrin in lipolysis ( dubuquoy et al . , 2011 ; he et al . , 2010 ; kershaw et al . was also confounded when pnpla3-deficient mice exhibited no detectable phenotype in lipid , lipoprotein , or energy metabolism ( basantani et al . overexpression of the i148 m variant of adiponutrin caused tg accumulation ( he et al . , 2010 ) , whereas overexpression of wild - type adiponutrin in the liver created no obvious phenotype ( he et al . , 2010 ) . in addition to classical lipolysis by extralysosomal neutral lipases , tgs and cholesteryl esters can also be hydrolyzed by lal . lal is thought to catabolize primarily lipoprotein - associated lipids subsequent to their receptor - mediated endocytosis and fusion with lysosomes . accordingly , the contribution of lal activity to lipolysis of intracellular lds was not considered relevant . given the lysosomal localization of lal , it was not intuitively obvious how lipids from lds would enter lysosomes . addressing this , singh and colleagues reported compelling evidence linking lipolysis to macroautophagy ( singh et al . , 2009a ) . macroautophagy is a lysosomal pathway that degrades superfluous or damaged organelles as well as cytoplasmic inclusions , such as misfolded protein aggregates ( levine and kroemer , 2008 ) . these cytoplasmic cargos are trapped inside double - membrane vesicles ( autophagosomes ) that ultimately fuse with lysosomes , where their contents are degraded . subsequently , lipids and aa are released into the cytosol and contribute to energy and aa supply in times of starvation . thus , along with lipolysis , macroautophagy is one of two conserved responses to organismal and cellular fasting . , 2009a ) found that , in addition to conventional neutral lipases , autophagy of lds is required for fasting - induced lipolysis in murine liver and cultured hepatocytes . they showed that recruitment of microtubule - associated protein 1 light chain 3 ( lc-3 ) and formation of a regional membrane through conjugation of autophagy - related protein 7 ( atg7 ) gives rise to double - membrane vesicles that engulf portions of cytoplasmic lds ( autolipophagosomes ) . the autolipophagosomes ultimately fuse with lysosomes , where their lipid content is degraded by lal . chronic fat feeding impairs the autophagic removal of lipid stores in the liver , prompting excessive hepatic lipid deposition . , 2010a ) strengthened these findings by showing that hepatic atg7 expression is severely impaired in ob / ob mice , contributing to the hepatosteatosis in these animals . while the effects of autophagy in genetically obese mice and in mice fed high - fat diets seem consistent , a role for autophagy in lipid metabolism of normal mice remains controversial . first , autophagy appears to be predominantly relevant in the liver after abnormally long fasting periods . normally , during shorter fasting periods the hepatic fat content increases due to the induction of adipose tissue lipolysis and increased fa supply to the liver . consistent with this prediction , hotamisligil and colleagues did not observe hepatic steatosis or changes in serum tg or fa levels in lean mice following sirna - mediated suppression of atg7 , arguing that lipoautophagy is not involved ( yang et al . , 2010a ) . second , hepatosteatosis as a result of atg7 deficiency was observed in some but not all studies . although atg7 deficiency leads to severe liver enlargement , it is controversial whether tgs accumulate . in fact , uchiyama and colleagues reported that hepatic atg7 deletion upon starvation inhibits ld formation both in vivo and in vitro , which leads to a lower hepatic tg content ( shibata et al . consistent with a role for lc3 in ld formation , rnai - mediated suppression of lc3-expression prevented ld formation in a panel of different ( hepatic and nonhepatic ) cell lines . interestingly , lc3 localized to the surface of lds , and the authors argued that lipidation of lc3 by phosphatidylethanolamine ( formation of lc3-ii ) , which is the initial step in autophagosome formation during autophagy , is also required for ld formation . a potential role of autophagy in lipogenesis but not in lipolysis is consistent with the finding that external administration of fas induces , rather than inhibits , autophagy when lds are formed in the fasting liver ( tang et al . , 2011 ) . a role for autophagy in lipogenesis also became evident from analysis of adipose tissue in atg7-deficient mice . assuming a lipolytic defect , we speculated that ablation of autophagy in adipocytes would result in an obese phenotype ( zechner and madeo , 2009 ) . in contrast , however , adipose - specific knockout of atg7 resulted in lean mice with reduced adipose mass , enhanced insulin sensitivity , and an elevated rate of -oxidation ( singh et al . , 2009b ) . the adipocytes contained smaller , multilocular lds and exhibited normal basal lipolysis . in line with this , inhibition of autophagy in cultured adipocytes using atg7 sirna blocked tg accumulation ( singh et al . these data argue for a currently poorly understood role of autophagy in the biogenesis of lds . interestingly , while adipose - specific atg7-deficient mice display reduced wat mass , bat mass increases ( baerga et al . , 2009 ; argued that inhibition of white adipocyte differentiation may lead to a defect in lipogenesis or that blocked autophagy may promote wat to bat transdifferentiation . in summary , autophagy may have pleiotropic roles in lipid metabolism depending on the cell- or tissue - type . in the liver , autophagy may contribute to lipolysis under a high - fat diet or prolonged fasting . , autophagy appears to be involved in adipocyte differentiation and lipogenesis but not in lipolysis . the role of autophagy in the breakdown of fat in other lipolytically active tissues , such as muscle , macrophages , and steroidogenic cells remains to be determined . textbook knowledge tells us that tgs are the most efficient way to store large amounts of fas as an energy reserve . consistent with this view , cellular lds were long seen as a relatively inert storage depot for fat in adipose tissue that is mobilized at times of increased energy demand . this view changed when it was realized that 1 ) lds are present in essentially all cell types , including those for which mere energy storage does not seem to be the main purpose ; 2 ) lds , particularly in nonadipose tissues , undergo very dynamic changes of formation and degradation ; and 3 ) lds represent a reservoir of bioactive lipids and lipid - derived hormones in adipose and nonadipose tissues . a role for neutral lipid metabolism in signaling gained substantial interest when it was noted that increased cellular tg concentrations are strongly associated with insulin resistance in skeletal muscle and liver ( cohen et al . the relatively inert nature of tgs makes it unlikely that they interfere directly with insulin signaling . the concept that tgs themselves are not the culprit was also supported by the so - called athletes ' paradox . endurance athletes accumulate more tgs in lds of skeletal myocytes than do untrained individuals , yet their muscle is highly insulin - sensitive . similarly , mice that lack atgl accumulate large amounts of fat in numerous tissues ( including skeletal muscle , cardiac muscle , liver , kidneys , and macrophages ) but exhibit increased insulin sensitivity ( haemmerle et al . , 2006 ; increased insulin sensitivity is observed despite the fact that atgl - deficiency leads to an insulin - secretion defect in pancreatic islets ( peyot et al . , 2009 ) . in humans , atgl - deficiency leads to neutral lipid storage disease with myopathy ( nlsdm ) , with a similar lipid phenotype as observed in atgl - deficient mice ( fischer et al . , 2007 ) . patients lacking the atgl coactivator cgi-58 not only develop neutral lipid storage disease but also exhibit a severe skin defect ( neutral lipid storage disease with ichthyosis , nlsdi ) ( lefvre et al . , 2001 ) . to date , defective pancreatic insulin production and alterations in insulin sensitivity have not been reported in patients with nlsdm or nlsdi . in conclusion , cellular tg content can be a marker of insulin resistance under certain physiological conditions but is not a regulator of insulin signaling . besides their powerful role as energy substrates and precursors of other lipids , fas or fa derivatives can bind to and activate members of the nuclear receptor family of transcription factors that control the expression of genes involved in lipid and energy homeostasis and inflammation . the ppar family consists of four members : ppar , ppar-1 and -2 , and ppar ( also designated ppar ) . ppar and ppar are highly expressed in oxidative tissues and regulate genes involved in substrate delivery , substrate oxidation , and oxidative phosphorylation ( oxphos ) . in contrast , ppar is more important in lipogenesis and lipid synthesis , with highest expression levels in wat . the full transcriptional activity of ppars requires the binding of cognate lipid ligands , heterodimerization with another nuclear receptor ( retinoid - x receptor , rxr ) , and interaction with a number of transcriptional coactivators , including ppar coactivator-1 ( pgc-1 ) . in addition to fas , other lipid ligands also have been described to activate ppars , such as acyl - coas , glycerol - phospholipids , and eicosanoids . fas involved in signaling originate from import of exogenous fas ( from circulating fa - albumin complexes or from lpl - mediated hydrolysis of plasma vldl and chylomicrons ) or from endogenous de novo synthesis . recently it was shown that neither source of fa can generate ppar ligands directly ; rather , a cycle of fa esterification and rehydrolysis is required ( haemmerle et al . , 2011 ) lipolysis - impaired atgl - deficient mice exhibit a severe defect in ppar signaling in oxidative tissues such as liver ( ong et al . , 2011 ) , macrophages ( chandak et al . , 2010 ) , and bat ( ahmadian et al . , 2011 ) . the most dramatic phenotype is observed in cardiac muscle ( haemmerle et al . , 2011 ) . the reduced expression of ppar target genes in atgl knockout animals causes severe mitochondrial dysfunction , decreased rates of substrate oxidation and oxphos , massive cardiac lipid accumulation , and lethal cardiomyopathy within a few months after birth . hsl deficiency is also associated with moderately decreased ppar target - gene expression but does not generate a comparable cardiac phenotype , indicating the specific importance of atgl activity in the generation of ppar ligands or ligand precursors . nlsdm or nlsdi in humans due to the deficiency of atgl or cgi-58 , respectively , may also lead to reduced ppar signaling and defective oxphos . although not proven experimentally , this assumption stems from the finding that nlsdm patients also develop systemic tg accumulation and cardiomyopathy . in many patients , this condition is lethal if they do not undergo heart transplantation ( hirano et al . , 2008 ) . importantly , at least in mice , the mitochondrial defect can be prevented by application of ppar activators , such as wy16453 or fenofibrate ( haemmerle et al . , 2011 ) . treatment of atgl - deficient mice leads to increased ppar signaling , disappearance of cardiac steatosis , and improved mitochondrial function and oxphos , as well as prolonged survival . whether pharmacological ppar activation will also be beneficial for patients with nlsdm is currently not known but remains a promising possibility . in addition to the instrumental role of atgl in ppar signaling , the enzyme may also affect ppar function . ( festuccia et al . , 2006 ) showed that rosiglitazone - mediated ppar activation and lipid accumulation are associated with increased lipolysis in wat of rats and that increased lipolysis was due to induction of atgl and mgl . although it seems counterintuitive that lipolysis is induced during increased tg synthesis , it is conceivable that this step is required to promote ppar-activated expression of lipogenic genes . moreover , the fact that hsl - deficiency also leads to downregulation of ppar target - gene expression in wat ( shen et al . , 2011 ; zimmermann et al . , 2003 it is well established that both plasma and cellular fa concentrations correlate positively with increased insulin resistance ( boden and shulman , 2002 ) . increased cellular concentrations of nonesterified fas , particularly palmitate , can drive the synthesis of lipotoxic lipids such as ceramides , which interfere with functional insulin signaling ( summers , 2010 ) . additionally , fas can directly or indirectly via increased production of reactive oxygen species activate redox - sensitive serine kinases , which , in turn , inactivate the insulin response ( vallerie and hotamisligil , 2010 ) . yet , not all fa species seem to have the same inhibitory effect on insulin signaling . whereas palmitate consistently decreases the insulin response , palmitoleate actually enhances the insulin signal in liver and muscle ( cao et al . , 2008 ) . its effects on insulin signaling in liver and muscle suggest that this fa is a lipokine with endocrine function . additionally , it is conceivable that hepatic palmitoleate also contributes to insulin sensitization in a paracrine fashion . whether lipolysis contributes to the generation of palmitoleate or other fas with a downstream effect on insulin signaling requires additional studies . atgl - deficient mice have low plasma fa concentrations and are highly insulin - sensitive , arguing for a protective role of reduced lipolysis and low fa levels . whether low plasma fas in hsl - deficient mice also affect insulin sensitivity is controversial ( mulder et al . , 2003 ; park et al . , 2005 ; voshol et al . , the potential of dgs to act as second messengers was discovered approximately 50 years ago when researchers realized that dgs affect many metabolic and mitogenic activities via activation of protein kinase - c ( pkc ) . metabolic regulation involves suppression of insulin signaling via phosphorylation of insulin receptor substrate-1 , leading to insulin resistance in muscle and liver ( samuel et al . , 2010 ) . only one dg stereoisomer , 1,2-diacyl - sn - glycerols ( 1,2-dgs ) , is able to activate pkcs , whereas the others , 1,3-diacyl - sn - glycerols ( 1,3-dgs ) and 2,3-diacyl - sn - glycerols ( 2,3-dgs ) , lack this bioactivity ( boni and rando , 1985 ) . 1,2-dgs activate conventional and novel pkc isoforms after recruitment of the enzymes to the plasma membrane by receptor of activated c kinase ( rack ) proteins ( turban and hajduch , 2011 ) . accordingly , both stereo - specific and location - specific preconditions are required for dgs to activate pkcs . classical signaling 1,2-dgs derive from phospholipase c ( plc)-mediated hydrolysis of phosphatidylinositol-4,5-phosphate in the plasma membrane . . both products of the enzymatic reaction , 1,2-dgs and inositol-1,4,5-phosphate ( ip3 ) , are potent second messengers . whereas 1,2-dgs remain plasma membrane - associated , ip3 dissociates and induces ca release from the er , which is required in addition to 1,2-dgs for activation of conventional pkcs . although plcs generate the correct stereoisomer ( 1,2-dgs ) in the correct cellular location for pkc activation , it remains controversial whether other cellular sources also contribute to the production of signaling 1,2-dgs . de novo synthesis via dual acylation of sn - glycerol-3-phosphate by acyl - coa glycerol-3-phosphate acyltransferases ( gpats ) and acyl - coa acylglycerol-3-phosphate acyltransferases ( agpats ) , and subsequent dephosphorylation of phosphatidic acid by phosphatidic acid phosphohydrolases ( papases ) also leads to the formation of 1,2-dgs ( figure 3 ) . however , this pathway of dg synthesis is restricted to er membranes . accordingly , a model proposing activation of pkc by er - associated 1,2-dgs needs to include pkc localization to the er or contact sites between the plasma membrane and the er membrane . the third potential source of dgs derives from the lipolysis of ld - associated tgs by atgl ( figure 3 ) . however , our unpublished observations showed that the enzyme preferentially hydrolyzes sn-1 and sn-2 ester bonds but not sn-3 esters . this indicates that atgl generates 1,3-dgs and 2,3-dgs but not 1,2-dgs . in accordance with the subsequent hydrolysis of 1,3-dgs and 2,3-dgs by hsl , this enzyme has a stereo - preference for the hydrolysis of fas in the sn-3 position of dgs ( rodriguez et al . , 2010 ) . in tgs , hsl preferably hydrolyses sn-1(3 ) ester bonds ( fredrikson and belfrage , 1983 ) . therefore , additionally , it is questionable whether ld - associated dgs would dissociate from lds to participate in the recruitment and activation of pkc at the plasma membrane . from all this , it seems unlikely that lipolytically generated dgs act as signaling mediators . in a recent review , shulman and colleagues ( samuel et al . , 2010 ) summarized numerous animal and human studies providing evidence that cellular dg concentrations account for the development of lipid - induced insulin resistance in type 2 diabetes , lipodystrophy , and other conditions . consistent with this hypothesis , mice lacking dg - kinase- , the major enzyme that inactivates the dg signal , have increased dg levels and increased insulin resistance ( chibalin et al . , 2008 ) . however , considering the structural complexity of dg species and their localization in different cellular compartments , a general correlation between total cellular dg concentrations and insulin resistance seems unlikely . this is supported by studies where increased total cellular dg concentrations in mutant mouse models were not associated with insulin resistance . for example , hsl - deficient mice accumulate large amounts of dgs in adipose and many nonadipose tissues due to defective dg catabolism . yet , most studies agree that this does not lead to pkc hyperactivation or a severely defective insulin response ( mulder et al . , 2003 ; park et al . , 2005 ; voshol et al . , similarly , cgi-58 silencing in the liver leads to increased dg levels , but normal ( chow diet ) or increased ( high - fat diet ) glucose tolerance and insulin sensitivity ( brown et al . , 2010 ) . taken together , determination of the specific 1,2-dg concentrations in the plasma membrane may provide a more reliable predictor for lipid - induced , pkc - mediated insulin resistance than total cellular dg concentrations . the signaling potential of mgs was recognized when it was found that the phospholipid - derived mg 2-ag activates cannabinoid receptors ( cbr ) , thereby regulating food intake , lipid metabolism , and energy homeostasis . the endocannabinoid system ( ecs ) refers to a group of neuromodulatory lipids ( endocannabinoids , ecs ) , two g protein - coupled receptors ( cbr1 and cbr2 ) , and enzymes involved in the synthesis and degradation of ecs ( di marzo , 2009 ) . the best - characterized ecs are n - arachidonoyl ethanolamine ( aea , anandamide ) and 2-ag . their biological effect is mimicked by -tetrahydrocannabinol ( thc ) , the major psychoactive component of marijuana . the ecs regulates a diverse spectrum of physiological processes , including motor function , pain , appetite , cognition , emotional behavior , and immunity . in the nervous system , 2-ag acts as a retrograde messenger , inhibiting presynaptic neurotransmitter release ( alger and kim , 2011 ) subsequently , 2-ag is internalized into the presynaptic terminal and inactivated by the mgl reaction , forming glycerol and arachidonic acid . the ecs is active in neurons and nonneuronal cells such as immune cells , hepatocytes , and adipocytes . treatment of obese patients with the cbr1-antagonist rimonabant ( christopoulou and kiortsis , 2011 ) and studies with animal models lacking cbr1 revealed that blockade of the ecs reduces food intake , decreases lipogenesis , and increases energy consumption . conversely , an overactive ecs has a central orexigenic effect and reduces energy expenditure , promoting lipid deposition in peripheral tissues like the liver and wat ( cota , 2008 ) . because of these biological effects obese patients may have an overactive ecs , which stimulates appetite and promotes lipid deposition ( perkins and davis , 2008 ) . it is generally assumed that signaling 2-ag originates from the degradation of glycerophospholipids containing arachidonic acid in the sn-2 position ( figure 3 ) . various isoforms of plc generate 1,2-dgs ( see above ) , which are subsequently hydrolyzed by dg lipase ( dagl ) to 2-ag . whether hsl also participates in the hydrolysis of plasma membrane - associated 1,2-dgs to generate 2-ag is not known . current evidence suggests that at least two isoforms of dagl ( dagl and dagl ) exist in the brain and liver ( bisogno et al . , 2003 ) . mice lacking dagl exhibit a substantial decrease in brain and spinal cord 2-ag levels , whereas dagl appears to be more important in peripheral tissues , such as the liver ( gao et al . , 2010 ) . whether the catabolism of arachidonic acid - containing tgs in lds by atgl and hsl also contributes to the cellular 2-ag and arachidonic acid pool is not known . recent studies using an mgl - specific small - molecule inhibitor ( jzl184 ) and mgl knockout mice provided compelling evidence that mgl is the major enzyme in the degradation of 2-ag and other mgs esterified with long - chain fas ( chanda et al . , 2010 ; schlosburg et al . , 2010 ; taschler et al . , animals lacking mgl or mice treated with jzl184 show abnormally high amounts of various mg species in the brain and peripheral tissues . in the brain , lack of mgl activity leads to a more than 20-fold increase in 2-ag , suggesting that mgl - deficiency could lead to hyperactivation of the ecs . indeed , jzl184 treatment of mice provoked cannabimimetic effects in mice , including analgesia , hypothermia , and hypomotility ( long et al . , however , genetic ablation of mgl in mice did not result in a hyperactive ecs or any obvious phenotype . this surprising observation was explained by desensitization of brain cbr1 leading to functional antagonism , and highlights the important role of 2-ag as a retrograde neurotransmitter ( chanda et al . , 2010 ; 2010 ) . obviously , increased brain 2-ag concentrations provoke counter - regulatory mechanisms similar to those observed when animals are chronically fed cbr agonists ( lichtman and martin , 2005 ) . although mgl - deficiency in mice does not produce cannabimimetic effects , the lack of mgl activity substantially affects lipolysis and metabolism in adipose tissue and nonadipose tissues ( taschler et al . , 2011 ) . mgl deficiency results in the accumulation of mgs and a reduction of circulating tg and glycerol levels in fasted animals . unexpectedly , and in contrast to the proposed role of the ecs in obesity - related metabolic diseases , mgl knockout mice exhibit improved insulin sensitivity and glucose tolerance when fed a high - fat diet . the cause for this finding may be complex , considering that mgl - deficiency is associated with desensitized cbrs . investigation of mice lacking mgl , specifically in the brain or peripheral tissues , should help to unravel the question of whether central or peripheral effects cause attenuation of insulin resistance . the first observation to indicate that lipolysis is linked to efficient cell - cycle progression was reported in the yeast s. cerevisiae . yeast expresses three tg lipases of the patatin domain - containing family termed tgl3 to 5 ( czabany et al . , 2007 ) . tgl4 is a functional ortholog of mammalian atgl ( kurat et al . , 2006 ) . deletion of tgl3 and tgl4 abolishes virtually all cellular tg lipase activity and causes a marked delay of entry into the cell division cycle of starved cells upon refeeding ( kurat et al . , 2009 ) . tgl4 is activated via phosphorylation by the cyclin - dependent kinase cdk1/cdc28 ( ortholog of mammalian cdc2 ) . this suggests that tg levels oscillate during the cell - division cycle to either deposit de novo synthesized fas in tgs or , conversely , to provide fas during phases of increased demand . tgl4 phosphorylation and activation occur at the g1/s transition of the cell - division cycle , which coincides with bud emergence and requires increased amounts of membrane lipids . pah1 phosphorylation and inactivation occur at the g2/m transition of the cell cycle , indicating that a window exists during the cell cycle in which both the initial step of lipogenesis and lipolysis may operate in parallel . this is feasible because both activities are confined to different organelles , namely the er and lds , respectively . the specific checkpoint proteins that regulate cell - cycle progression in response to lipolysis are currently unknown . recent evidence shows that the synthesis of phosphatidylinositol ( pi ) , a precursor for signaling molecules in cell - cycle regulation in yeast ( e.g. , ip3 and inositol - containing ceramides ) , strongly depends on intact lipolysis ( gaspar et al . , 2011 ) . thus , cell cycle - regulated tg lipolysis may provide critical precursors for signaling molecules for cell division . a highly interesting study recently demonstrated that mgl promotes the oncogenic properties of mammalian cancer cells ( nomura et al . , 2010 ) . the authors demonstrated that overexpression or disruption of mgl activity increased or decreased , respectively , the proliferation of cancer cells , and that mgl is highly expressed in aggressive tumor cell lines or primary tumors . the study also provided evidence that mgl influences tumor proliferation by metabolic effects rather than by cbr - dependent mechanisms . growth of tumor cells in response to mgl - knockdown was not impaired after addition of exogenous nonesterified fas or in mice fed a high - fat diet . this led to the conclusion that mgl affects the concentration of fa - derived tumorigenic lipid metabolites , such as lpa and pge2 . although the involved lipid signal(s ) requires identification , these studies clearly designate mgl as an interesting target for cancer therapy . lipolytic signaling may also be causally involved in the pathogenesis of cancer - associated cachexia ( cac ) . in a recent study , das et al . ( das et al . , 2011 ) demonstrated that atgl - deficient mice are protected against tumor - induced loss of adipose tissue and skeletal muscle . atgl - deficient mice maintained body weight and composition despite increased circulating factors that induce lipolysis , muscle proteolysis , and apoptosis ( e.g. , tnf , interleukin-6 , and zinc--glycoprotein 1 ) . this suggests that lipolysis is integrated in a signal transduction network that eventually leads to the loss of adipose tissue and muscle . this view is also consistent with the observation that the activity of lipolytic enzymes and release of fas and glycerol are increased in adipose tissue of cancer patients with cachexia ( agustsson et al . , 2007 ; das et al . , 2011 ; rydn et al . , it is also unclear whether the signal originates from lipolysis in one tissue ( such as adipose tissue ) and promotes wasting in an endocrine manner or whether wasting requires autonomous lipolysis in all tissues that are affected by wasting . although the underlying mechanism is not yet defined , this study suggests that inhibition of lipolysis may help to prevent cachexia in patients with cancer or other chronic diseases . recent discoveries of enzymes and regulatory factors have led to a revision of our perception of lipolysis . additionally , we have just begun to address the role of lipolytic products and intermediates in cellular signaling . important topics for future investigations include : 1 ) better understanding of the biochemical factors and processes that coordinately regulate the lipolytic machinery in response to hormonal activators and inhibitors , 2 ) the physiological function of lipolysis in numerous nonadipose tissues and the tissue - specific differences in lipolytic mechanisms , and 3 ) the characterization of lipolytic signals and the molecular mechanisms of their effects on gene transcription , the cell cycle , and cell growth . the recent examples of lipases affecting tumor proliferation or cancer - associated cachexia emphasize the potential importance of lipolysis in human disease .

the usefulness of basic cardiopulmonary resuscitation ( cpr ) training in school systems has been questioned , considering that young students may not have the physical or cognitive skills required to perform complex tasks correctly . in the study conducted by fleishhackl and coworkers , students as young as 9 years were able to successfully and effectively learn basic cpr skills , including automated external defibrillator deployment , correct recovery position , and emergency calling . as in adults , physical strength may limit the depth of chest compressions and ventilation volumes given by younger individuals with low body mass index ; however , skill retention is good . training all persons across an entire community in cpr may have a logarithmic improvement in survival rates for out - of - hospital cardiac arrest because bystanders , usually family members , are more likely to know cpr and can perform it immediately , when it is physiologically most effective . training captured audiences of trainees , such as the entire work - force of the community or the local school system , are excellent mechanisms to help achieve that goal . in addition to better retention with new half hour training kits , a multiplier effect can be achieved through school children . in addition , early training not only sets the stage for subsequent training and better retention , but it also reinforces the concept of a social obligation to help others .

the prospective investigation conducted by fleischhackl and coworkers , reported in the previous issue of critical care , sets out to determine whether young students have the physical and cognitive skills to implement cardiopulmonary resuscitation ( cpr ) . in this investigation , the average time from the last class of cpr instruction to the evaluation session was 120 days . it is not clear whether such a large gap in time between initial instruction and skills testing may have affected testing performance , except that good performance could indicate good retention . students tested also included those with special learning needs , which may skew the examination results in an unfavorable direction . nevertheless , the outcomes were generally very good . as the investigators demonstrated , students as young as 9 years are able to effectively learn cpr skills , including automated external defibrillator deployment , correct recovery position , and emergency calling . as in adults , physical strength may have limited the depth of chest compressions and ventilation volumes , but perhaps the key finding was that skills retention was good . these findings are consistent with other studies in which none of the students aged 9 to 10 years could compress the chest to the depth recommend by the guidelines , but 45% of students aged 13 to 14 years old could . studies also have found that with retraining , cpr performance can improve in school - aged children . although the study by fleischhackl and coworkers did not specifically address this retraining , it would have been interesting to know how well these students would have retained their learned skills several months later , because it is well known that cpr skills rapidly deteriorate after initial training . intuitively , the higher the number of persons trained in cpr skills in a given community , the more frequently it is performed . for example , in los angeles , where the relative percentage of citizens trained in cpr is estimated to be relatively low , only a small percentage of bystanders performed cpr and just 1.4% of out - of - hospital cardiac arrest victims survived . however , 10-fold improvements in survival rates ( > 15% ) have been reported in seattle , where the frequency of bystander - performed cpr is one of the highest in the nation . in essence , one of the most effective means of improving surviving for out - of - hospital cardiac arrest is to attempt to train all individuals across a given community , and experience has shown that having captive audiences for training , such as in school systems , is a major component . when closely examining such numbers , the survival improvements are not linear but may actually be logarithmic . young children between the ages of 10 and 14 years only account for approximately 15% of the population in the usa , and they are not in the main target age group for cpr except for perhaps drowning incidents . however , there are multiple benefits from teaching school - aged children cpr beside the concept of capturing entire generations of cpr - trained citizens . children older than 10 years of age are teachable and capable of abstract thought , and most have the coordination and physical strength to perform cpr . the cpr kits that are currently being promoted by the american heart association are designed to train individuals who are as young as 8 years old in less than a half hour . it has been demonstrated that using such a kit that contains a self - instructional video and an inflatable manikin not only promotes retention but also has a multiplier effect . one study found that distributing cpr training kits to students aged 12 to 14 years resulted in another 2.5 persons trained per student . furthermore , early training can lay the foundation for a sense of social obligation and reinforce cpr knowledge and follow - up training , so that by the time a student graduates from high school cpr skills are well engrained and can easily called upon in an emergency situation . if government agencies enforced mandatory age - appropriate cpr and first aid training in schools , similar to how schools practice evacuation and file drills , then one could only imagine the impact this could have on the number of individuals capable of intervening in out - of - hospital cardiac arrest and , in turn , the number of lives saved .

background and aims : earlier studies have shown that the type of laryngoscope blade influences the degree of hemodynamic response to endotracheal intubation . the aim of the study was to evaluate the hemodynamic response to oral endotracheal intubation with c - mac laryngoscopy and mccoy laryngoscopy compared to that of macintosh laryngoscopy in adult patients under general anesthesia.material and methods : this is a prospective randomized parallel group study . ninety american society of anesthesiologists i patients were randomly allotted into three groups . group a macintosh laryngoscopy ( control group ) . group b laryngoscopy with mccoy laryngoscope . group c laryngoscopy with c - mac video laryngoscope . heart rate ( hr ) , systolic blood pressure ( sbp ) , diastolic blood pressure ( dbp ) , and mean arterial pressure ( map ) were monitored at baseline ( just before induction ) , just before intubation ( t0 ) , 1 min ( t1 ) , 3 min ( t3 ) , 5 min ( t5 ) , and 10 min ( t10 ) after intubation . intergroup comparison of study parameters was done by unpaired sample t - test for normal data and mann - whitney u - test for skewed data . for within - group comparison , the repeated measures of anova for normal data and friedman followed by wilcoxon signed rank test for skewed data were performed.results:in c - mac group , the hr was significantly higher than the macintosh group at 3 min after intubation , whereas sbp , dbp , and map were significantly higher at 1 min . mccoy group showed a similar response compared to macintosh group at all time intervals.conclusion:c-mac video laryngoscope has a comparatively greater hemodynamic response than macintosh laryngoscope .

it has also been shown that the type of laryngoscopic blade influences the degree of hemodynamic response . videolaryngoscopes have shown varying results such as pentax aws ( pentax corporation , tokyo , japan ) has an attenuated response , whereas glidescope ( saturn biomedical system inc . , burnbaby , canada ) has shown similar hemodynamic response compared to macintosh laryngoscopy and endotracheal intubation . however , there are no studies evaluating the hemodynamic response of c - mac video laryngoscope ( karl storz gmbh & co. kg , tuttlingen , germany ) for endotracheal intubation . hence , we compared the hemodynamic response to oral endotracheal intubation using mccoy and c - mac laryngoscopy with that of conventional macintosh laryngoscopy in adult patients posted for elective surgery under general anesthesia . one recent study has shown that c - mac videolaryngoscopy needed less duration of laryngoscopy for successful intubation compared to macintosh laryngoscopy even in lateral position . duration of laryngoscopy is an expected variable to affect the degree of hemodynamic response to endotracheal intubation . hence , we hypothesized that the mccoy or c - mac laryngoscopy produces an attenuated hemodynamic response to laryngoscopy and oral endotracheal intubation compared to macintosh laryngoscopy . with the institute ethics committee 's approval ( csp - med/14/feb/12/34 dated 13 - 03 - 2014 ) and informed consent from all patients , this prospective randomized parallel group study was carried out . the procedures followed were in accordance with the ethical standards of the institutional research ethics committee standards and with that of the helsinki declaration of 1975 , as revised in 2000 . we included ninety american society of anesthesiologists ( asa ) i patients of 1840 years of age posted for elective surgery under general anesthesia . pregnant women , patients undergoing emergency surgery and those with airway abnormalities , and anticipated difficult airway ( mallampati class iii and iv , thyromental distance < 6 cm , inter - incisor distance <3 cm , and cervical instability ) were excluded from the study . in addition , the patients in whom laryngoscopy lasted for more than 30 s and if more than one intubation attempt was needed were also excluded . the study population was randomly allocated into three groups , namely group a ( n = 30 ) laryngoscopy with macintosh laryngoscope ( blade size 3 ) control group , group b ( n = 30 ) laryngoscopy with mccoy laryngoscope ( blade size 3 ) , and group c ( n = 30 ) laryngoscopy with c - mac video laryngoscope ( d - blade ) . randomization was done by computer - generated random numbers and concealed by sealed envelope technique . this was done by a separate anesthesiologist who was not involved in performing laryngoscopy or data collection during the study . laryngoscopy and intubation were performed by a single senior anesthesiologist in all cases , who was familiar and experienced in intubation using both mccoy and c - mac laryngoscope . endotracheal tubes of size 8.5 mm i d in males and 7.5 mm i d in females were used . the endotracheal tubes were loaded with stylet shaped in a hockey stick fashion in all patients . standard monitoring was done with electrocardiogram ii and v leads , noninvasive blood pressure , and pulse oximetry ( phillips intellivue mp50 . , philips healthcare , netherlands ) . after preoxygenation for 3 min with 100% oxygen , anesthesia was induced with midazolam 1 mg , fentanyl 2 mcg / kg , and thiopentone 5 mg / kg intravenously , and maintained with sevoflurane 2% in 100% oxygen . after checking for mask ventilation vecuronium 0.1 mg / kg was administered intravenously . at the end of 3 min , etco2 was maintained below 40 mmhg to avoid the effects of hypercarbia on the hemodynamic variables during the study period . no other medications were administered or procedures performed during the 10 min data collection period after endotracheal intubation . the study parameters monitored were heart rate ( hr ) , systolic blood pressure ( sbp ) , diastolic blood pressure ( dbp ) , and mean arterial pressure ( map ) . the above parameters are measured at the following intervals : baseline ( bl before induction ) , just before intubation ( t0 ) , and 1 min ( t1 ) , 3 min ( t3 ) , 5 min ( t5 ) , and 10 min ( t10 ) after intubation . the first reading of all the parameters after connecting the monitor inside the operating room and before preoxygenation was taken as bl reading . all the study parameters were collected and documented by the same observer who is blinded for the type of laryngoscope used . other parameters measured are duration of laryngoscopy with a stop clock by an independent observer . duration of laryngoscopy was defined as the time from the insertion of laryngoscope into the oropharynx till the appearance of first etco2 curve . intubation response was defined as : the hemodynamic changes in terms of hr , sbp , dbp , and map that occurs immediately and within 10 min after oral endotracheal intubation . significant hemodynamic response that would warrant intervention is defined in the study as a change in hr or blood pressure with a 20% change from bl . decrease in blood pressure or hr was treated with injection ephedrine 6 mg intravenous boluses . sample size calculation was done based on a pilot study done by us prior to the study with ten patients in each group comparing the hemodynamic response to laryngoscopy and oral endotracheal intubation with macintosh , mccoy , and c - mac videolaryngoscope . comparison of macintosh and c - mac group needed a sample size of 25 in each group to detect an effect size of 10 mmhg in the systolic pressure at a standard deviation ( sd ) of 12.8 with a power of 80% and alpha error of 0.05 whereas comparison of macintosh and mccoy group needed a sample size of about 23 patients in each group to detect an effect size of 13.6 mmhg in systolic pressure at a sd of 16 with a power of 80% and alpha error of 0.05 . sample size calculation based on the data of previous study comparing the hemodynamic response with macintosh and mccoy laryngoscopy also arrived at a similar sample size . the collected data were analyzed with spss for windows , version 16.0 ( spss inc . , chicago , il , usa ) . to describe about the data descriptive statistics , mean and hr , dbp , and demographic data had a normal distribution whereas sbp and map had a skewed distribution . the demographic data were analyzed by one - way analysis of variance . to find the significant difference between the bivariate samples in independent groups , the unpaired sample t - test was used for normal data and mann - whitney u - test for skewed data . for the repeated measures , the repeated measures of anova with adjustment for multiple comparisons to control the type i error , with bonferroni test was used for normal data and friedman followed by wilcoxon signed rank test for skewed data . in all the above statistical tools , the p < 0.05 was considered significant level . those patients who needed intervention with ephedrine were excluded from the study , since the subsequent readings would be affected by its administration . the demographic data in terms of age , weight , and height was similar in all groups [ table 1 ] . there was no statistically significant difference in the bl values of all the study parameters among the three groups . comparison of demographic data among groups on studying each group separately , in group a ( macintosh ) , there was no statistically significant increase in any of the study parameters at all study intervals after intubation compared to the bl value [ tables 2 and 3 ] . whereas in group b ( mccoy ) , there was a statistically significant increase in hr response at 1 min interval ( t1 ) from bl value with p = 0.003 , and at other time intervals , there was no significant rise from bl in any of the study parameters [ tables 2 and 3 ] . comparison of mean heart rate and systolic blood pressure of group b and group c with group a comparison of mean diastolic blood pressure and mean arterial pressure in group b and group c with group a there was a statistically significant increase in the hr at 1 min ( t1 ) ( p = 0.001 ) and 3 min ( t3 ) ( p = 0.004 ) interval from the bl hr in group c. whereas there was a statistically significant increase in dbp ( p = 0.01 ) and map ( p = 0.03 ) at t1 interval compared to the bl in group c ( c - mac ) . at other time intervals , there is no rise but a significant fall in the values compared to the bl value [ tables 2 and 3 ] . table 2 compares the changes in hr and sbp of group b and group c with that of group a. the difference in hr at 3 min interval ( t3 ) after intubation between group a and group c was statistically significant t(58 ) = 2.782 , p = 0.008 . the hr was significantly higher in group c compared with group a at 3 min ( t3 ) interval . at other time intervals , there is no significant difference in hr on comparing group b and group c with that of group a. there was a significant difference in sbp observed when comparing group a with group c u = 294 , p = 0.01 , and r = 0.42 at 1 min ( t1 ) after intubation . at other intervals , there was no statistically significant difference in sbp on comparing group b and group c with that of group a. table 3 compares the changes in mean dbp and mean artery pressure of group b and group c with that of group a. there is a statistically significant difference in dbp observed between group a and group c , t(58 ) = 2.301 , p = 0.03 at 1 min interval ( t1 ) after intubation . at other time intervals , there are no statistically significant changes in dbp on comparing group b and group c with that of group a. there is a statistically significant difference in mean artery pressure observed on comparing group a with group c ; u = 320 , p = 0.04 , and r = 0.35 at 1 min interval ( t1 ) after intubation . at other intervals , there are no statistically significant changes in map on comparing group b and group c with that of group a. we found that c - mac laryngoscope has higher hemodynamic response to tracheal intubation compared to conventional macintosh laryngoscopy and intubation in these asa i patients . while comparing the macintosh with mccoy laryngoscopy , we found that there is no significant difference in terms of hemodynamic response to orotracheal intubation between the two laryngoscopic methods . some of the previous studies have shown that there is no difference in hemodynamic response between mccoy laryngoscope and conventional macintosh laryngoscopy . one study comparing mccoy and macintosh laryngoscope found that the mccoy group had less hemodynamic response when fentanyl was not included as premedicant , but there was no difference when fentanyl was used as premedicant . in our study , both the macintosh and mccoy group did not show any significant increase in the study parameters , but there was a decreasing trend after intubation . this can be attributed to the difference in the inclusion criteria compared to other studies , namely the asa status and the age group . unlike earlier studies , we included only the asa i patients belonging to 1840 years of age group in our study . a recently published study compares the hemodynamic response to endotracheal intubation with macintosh and c - mac videolaryngoscope in cardiac surgical patients . they found no significant change between the two groups in terms of the hemodynamic response to laryngoscopy and endotracheal intubation . this particular study has been done in heterogeneous cardiac surgical patients with varied cardiac pathology coming for cabg , valvular surgeries , etc . the patients were on drugs such as beta blockers which itself would have influenced the degree of hemodynamic response to endotracheal intubation , while our study has shown that c - mac has an increased hemodynamic response to intubation compared to macintosh laryngoscopy and endotracheal intubation . the difference in the study population would have contributed to the difference in the outcome between the two studies . the major limitation was the difficulty in blinding with this study design which could have led to a potential bias in the study . however , we ensured that a separate anesthesiologist not knowing the type of laryngoscopy is involved in data collection . secondly , the learning curve of the new technique of laryngoscopy with c - mac videolaryngoscope has not been established and this would have affected the hemodynamic response to endotracheal intubation . the authors did this to avoid the potential bias that would happen when a wide variable group such as asa ii patients was included . such patients may have conditions such as hypertension , diabetes , and hypothyroidism , which can affect the hemodynamic response to oral endotracheal intubation . furthermore , conducting the entire study in controlled hypertensives will be technically difficult to recruit patients as well as to standardize the confounding factors such as drug therapy . moreover , the authors felt that attenuation of hemodynamic response to endotracheal intubation is also important in these asa i patients while providing general anesthesia . our study showed that mccoy and macintosh laryngoscopies have similar hemodynamic response to direct laryngoscopy and endotracheal intubation , but c - mac video laryngoscopy and endotracheal intubation have an increased hemodynamic response than conventional macintosh laryngoscopy and intubation in patients undergoing general anesthesia .

every infant born in the us is now screened for up to 42 rare genetic disorders called " inborn errors of metabolism " . the screening method is based on tandem mass spectrometry and quantifies acylcarnitines as a screen for organic acidemias and also measures amino acids . all states also perform enzymatic testing for carbohydrate disorders such as galactosemia . because the results can be non - specific , follow - up testing of positive results is required using a more definitive method . the present report describes the " urease " method of sample preparation for inborn error screening . crystalline urease enzyme is used to remove urea from body fluids which permits most other water - soluble metabolites to be dehydrated and derivatized for gas chromatography in a single procedure . dehydration by evaporation in a nitrogen stream is facilitated by adding acetonitrile and methylene chloride . then , trimethylsilylation takes place in the presence of a unique catalyst , triethylammonium trifluoroacetate . automated injection and chromatography is followed by macro - driven custom quantification of 192 metabolites and semi - quantification of every major component using specialized libraries of mass spectra of tms derivatized biological compounds . the analysis may be performed on the widely - used chemstation platform using the macros and libraries available from the author . in our laboratory , over 16,000 patient samples have been analyzed using the method with a diagnostic yield of about 17%--that is , 17% of the samples results reveal findings that should be acted upon by the ordering physician . included in these are over 180 confirmed inborn errors , of which about 38% could not have been diagnosed using previous methods .

take a maximum aliquot of 13 ml of sample and store it at -20 c in a labeled conical centrifuge tube . measure and record the optical density of the sample by placing a couple of drops of urine in the refractometer . filter the sample through a 0.2 um filter . measure out the volume of sample below according to the optical density 1.000 - 1.0091.00 ml1.010 - 1.0190.50 ml1.020 - 1.0500.25 ml + 0.25 ml h2o the specified volume is then transferred to a reactivial containing the following internal standards : 500 nanomoles ( nmoles ) 3 creatine ; 10 nmoles d3 methylmalonic acid ; 100 nmoles each of the following c3 lactate , c3 pyruvate , c2 n glycine , d3 serine , d5 phenylalanine , d11 hexanoylglycine , n2 orotate , d4 sebacic acid , c6 glucose , d6 inositol and d5 tryptophan . 20 microliters ( l ) of a 7.5 units/l solution of urease ( calzyme laboratories catalog no . 116a0100 ) is added to the sample , which is then flushed and sealed under co2 through an inert septum . the sample is held at 37c for 30 minutes with more carbon dioxide gas added at 15 minute intervals to maintain pressure . 20 l more of the urease solution is added , the vial is flushed with carbon dioxide , and the sample maintained at 37c for another 15 minutes . 500 l of 30:70 acetone : methanol is added , the rubber septum is replaced with a teflon coated septum , and the sample is chilled at -20 c for 15 minutes . solids are removed by centrifugation at 1500 rpm x 10 minutes then decanted into a clean 2.0 cc reactivial ( supelco / sigma ) add triethylammonium trifluoroacetate ( tea / tfa ) ( sigma ) as follows : 20 l for 1.00 ml samples40 l for 0.5 ml , or less , samples 20 l for 1.00 ml samples 40 l for 0.5 ml , or less , samples top off with acetonitrile and place under a nitrogen stream at 70 c until constant volume is achieved ( tea / tfa will remain ) ( ~ 15 minutes ) . repeat step 13 , up to 4 times until a precipitate forms ( ~ 10 minutes each ) . top off with methylene chloride , being careful of boil over , and dry ( ~ 4:00 minutes ) . add mstfa ( n - methyl - n - trimethylsilyltrifluoroacetamide ) ( thermal scientific ) at the following rates : 150 l for 1.00 ml samples200 l for 0.50 ml , or less , samples 150 l for 1.00 ml samples 200 l for 0.50 ml , or less , samples cap under a nitrogen atmosphere and incubate at 70 c for 1 hour . transfer to microvials , under a nitrogen atmosphere , for analysis on gas chromatograph /mass spectrometer . place microvials for automated injection by the agilent 5975 gc / ms : instrument temperatures are : injector 200 c , interface 250 c , oven 80 c for 1 minute ; ramp at 4 c / minute 80 - 130 c , ramp at 6 c/ minute 130 - 200 c , ramp at 12 c/ minute 200 - 285 c , hold for 10 minutes . . mass spec : source 230 c , quad 150 c , scan 50 - 650 amu at 2.46 scans / sec . take a maximum aliquot of 13 ml of sample and store it at -20 c in a labeled conical centrifuge tube . measure and record the optical density of the sample by placing a couple of drops of urine in the refractometer . filter the sample through a 0.2 um filter . measure out the volume of sample below according to the optical density 1.000 - 1.0091.00 ml1.010 - 1.0190.50 ml1.020 - 1.0500.25 ml + 0.25 ml h2o the specified volume is then transferred to a reactivial containing the following internal standards : 500 nanomoles ( nmoles ) 3 creatine ; 10 nmoles d3 methylmalonic acid ; 100 nmoles each of the following c3 lactate , c3 pyruvate , c2 n glycine , d3 serine , d5 phenylalanine , d11 hexanoylglycine , n2 orotate , d4 sebacic acid , c6 glucose , d6 inositol and d5 tryptophan . 20 microliters ( l ) of a 7.5 units/l solution of urease ( calzyme laboratories catalog no . 116a0100 ) is added to the sample , which is then flushed and sealed under co2 through an inert septum . the sample is held at 37c for 30 minutes with more carbon dioxide gas added at 15 minute intervals to maintain pressure . 20 l more of the urease solution is added , the vial is flushed with carbon dioxide , and the sample maintained at 37c for another 15 minutes . 500 l of 30:70 acetone : methanol is added , the rubber septum is replaced with a teflon coated septum , and the sample is chilled at -20 c for 15 minutes . solids are removed by centrifugation at 1500 rpm x 10 minutes then decanted into a clean 2.0 cc reactivial ( supelco / sigma ) add triethylammonium trifluoroacetate ( tea / tfa ) ( sigma ) as follows : 20 l for 1.00 ml samples40 l for 0.5 ml , or less , samples 20 l for 1.00 ml samples 40 l for 0.5 ml , or less , samples top off with acetonitrile and place under a nitrogen stream at 70 c until constant volume is achieved ( tea / tfa will remain ) ( ~ 15 minutes ) . repeat step 13 , up to 4 times until a precipitate forms ( ~ 10 minutes each ) . top off with methylene chloride , being careful of boil over , and dry ( ~ 4:00 minutes ) . add mstfa ( n - methyl - n - trimethylsilyltrifluoroacetamide ) ( thermal scientific ) at the following rates : 150 l for 1.00 ml samples200 l for 0.50 ml , or less , samples 150 l for 1.00 ml samples 200 l for 0.50 ml , or less , samples cap under a nitrogen atmosphere and incubate at 70 c for 1 hour . transfer to microvials , under a nitrogen atmosphere , for analysis on gas chromatograph /mass spectrometer . place microvials for automated injection by the agilent 5975 gc / ms : instrument temperatures are : injector 200 c , interface 250 c , oven 80 c for 1 minute ; ramp at 4 c / minute 80 - 130 c , ramp at 6 c/ minute 130 - 200 c , ramp at 12 c/ minute 200 - 285 c , hold for 10 minutes . column : 25 m , 320 micron i d , 0.5 micron film thickness db-5 . mass spec : source 230 c , quad 150 c , scan 50 - 650 amu at 2.46 scans / sec . the urease method ( 1 ) has been cited 62 times in the medical literature with various modifications . matsumoto 's group ( 2,3 ) simplified the procedure for high - throughput neonatal screening and reported the results from 16000 patients . kuhara and others ( 4 - 7 ) have reported the use of the method in several cases of inborn error diagnosis and follow - up . rhead ( 8) also confirmed the method 's utility for clinical diagnosis and follow - up of inborn errors . the method has been applied to urine of bears , knock - out mice , elephants and homogenates of whole fruit flies and their larvae ( 9 ) . culture media from cryptococcus before and after site - directed mutagenesis were also analyzed , without the urease step ( 10 ) . the method has been applied to human nutritional assessment in medical students , down syndrome patients and demented elderly veterans after loading the subjects with oral doses of the amino acids tryptophan , methionine and isoleucine ( 11 ) . all eight b - vitamins were assessed by quantifying the breakdown products of the three amino acids which , among them , require all 8 vitamins at some point in their degradation . the toxic effects of pharmaceuticals and their mitigation by vitamin supplementation has been reported ( 12 ) . amniotic fluid samples from normal and down syndrome pregnancies were analyzed and reported ( 13 - 15 ) . the metabolic screening lab is a clia - licensed clinical laboratory owned by saint louis university , a non - profit corporation . dr . shoemaker does not directly profit from laboratory revenues , but may benefit indirectly from laboratory productivity . none of the methods , techniques , results , normal ranges , macros , software , libraries or conclusions are considered proprietary and are freely available to interested parties .

background : long wait time interval in emergency department ( ed ) of hospitals , from the patients point of view in ed is a major problem causing patients dissatisfaction and may result increasing in patient morbidity and indirectly nurses dissatisfaction . evaluation of wait time intervals in ed and giving nursing feedback may improve the quality of services , as well as patient satisfaction . the present study was designed to investigate the effect of nursing triage on receiving treatment of wait time interval and satisfaction of the patients referring to ed in shahid rajaee hospital.materials and methods : this study was conducted on patients those referring to shahid rajaee hospital in karaj , iran employing quasi experimental design d ividing in two experiment and control groups during 2009.this is a quasi - experimental study of which the data were collected by standard questionnaire covering patient satisfaction and measuring wait time . t - test , mann - whitney and frequency analysis were used to evaluate the effect of triage on wait time from receiving treatment services and patients satisfaction.results:the findings showed that there was a significant difference between experiment and control groups regarding wait time from receiving treatment services and patients satisfaction.conclusions:triage could significantly reduce the wait time interval between patients entrance to ed to receive treatment services and enhance patients satisfaction . it may help nursing in emergency ward to have better performance and indirectly their satisfaction .

the patients referring to hospitals emergency department ( ed ) needing immediate care comprise 78% of all patients . minutes or even the seconds are crucial for these patients since , 75 - 85% of mortality occurs in the first 20 min post - events ( such a s head injury ) . most of the injuries either manage or progress within the first 10 min when major decisions are made . time limit , high number of clients , various clients , lack of background information about them , limitation of diagnostic interventions , and the urgency of selecting related treatment are amongst ed features . from the patients viewpoint , the , long waiting time interval resulting in patients dissatisfaction with emergency services is a major problem in hospitals eds , as well as their pre - hospital phases . basically , increase of wait time is one of the major reasons for the crowd in eds resulting in patients leave with no physicians evaluation , a delay in treatment , patients dissatisfaction and jeopardizing their lives . on the other hand , a decrease in wait time to receive emergency services brings about on time treatment , a decrease in hospitalization time interval , lower treatment costs and saving in hospital resources . meanwhile , the most important criterion used to evaluate eds is patients waiting time to receive diagnostic and treatment services . triage is one that is the system , which can be easily administrated and manage the time of diagnosis and treatment with regard to patients status . triage is a process that an emergency disease or an injury is classified for the patients referring to eds in order to provide an appropriate level of treatment and care , be prioritized and transferred in the shortest possible time . most of times , nurses are the first health staffs admitting the patients to diagnose their problem and administrate emergency care for them . previous studies have been already conducted on patients time waste in eds with regard to their satisfaction in iran , but not on the effect of triage on wait time decrease in eds . accordingly , there is a few studies in iran regarding waiting time and discharge time in emergency department ; for instance , study in the kashani hospital in esfahan has been shown that the average time for patients to complete the discharge process in ed was 4.9 h ; the other in kerman has showed that mean for wait time in ed in bahonar hospital was about 7 h. both studies indicated that long wait time directly affect patients dissatisfaction . however , the effect of triage on wait time and discharge time did not measure in these studies . evaluation of ed wait time interval and patients point of view may improve the quality of emergency care at ed . due to feasibility and a lack of similar study in shahid rajaee hospital in karaj , the present study was designed to investigate the effect of triage on wait time to receive treatment services and patients satisfaction those referring to karaj ed . this is a quasi - experimental study , which was conducted on the patients referring to ed of shahid rajaee hospital in karaj during 2009 . experimental condition was the triage as intervention , which was made on cases group vs. control group that did not have triage ( see below ) . according to quasi - experimental design , wait time and patient satisfaction were as dependent variables and then triage was manipulated to check its effect on both groups considering wait time and patient satisfaction . its emergency department provides both inpatient and outpatient services for internal cardiac and traumatic patients care . five nurses work in morning shift , four in evening shift , and four nurses in each night shift . considering previous similar studies and due to its feasibility , the samples were considered as 600 , by means of 300 cases and 300 in experiment group . all patients in experiment group were triaged at the time of their entrance vs. control group that did not triage . patients those come to the ed were included in the study ; except those needing cardiopulmonary resuscitation ( cpr ) at the time of their arrival ; and those who had no vital signs , which was no need for their triage , were left out from the study . a questionnaire including demographic questions , as well as two sections of patient satisfaction and time measurement was used to collect the data . age , sex , referral route and time measurement included information concerning time of arrival and time of the first visit by a physician were considered . satisfaction measurement section contained 11 questions covering patients satisfaction with ed services concerning personnel 's behavior in admission , existence of facilities and equipments , and ed staffs reaction time to start emergency services . it is important to beer in mind that the data collection tool is a national and standard questionnaire that had been made by in ministry of health and medical education and had been used already and confirmed regarding its validity and reliability . the principal researcher filled the questionnaire after getting consent from the clients with no intervention in their triage . the data were collected through interview with patients , or their accompanying if patients are not quite alert and conscious . using random block sampling method , starting the first day of the study conducting , the principle investigator attends at ed first in morning following in evening and night shift , respectively . questionnaire of satisfaction was filled at the time of discharge or their transferring to ward for inpatients cases ; and at the time of leaving ed for outpatients . then , the data of wait time for the first visit in ed were collected and satisfaction with the services was sought and then triage was administrated . after primary triage of the patients , they were classified and transferred to ed to be visited by a general physician and to receive related care . there were two physicians present in each shift ( one for class one referrals and one for outpatients of class two and three ) . wait time was measured from early arrival of the patients to triage room ( prioritizing with triage system ) to the general physicians visit . the data were analyzed by spss version 13 ( spss , chicago , il ) , as a means of t - test to evaluate the effect of triage on wait time to receive treatment services in experiment vs. control group . mann whitney test was used to compare patients satisfaction to test the effect of triage in tow different groups . the frequency of sex ( male and female ) , education level ( illiterate , primary school , secondary school , high school , associate degree , bachelor 's degree and over ) marital status ( single , married , divorces ) and referral times to ed ( first , second or over ) was compared in both experiment and control groups . the study was approved by the ethics committee of university of social welfare and rehabilitation , tehran , iran . the interviewees then signed an informed consent form or verbally consented to participate in the study . the study was approved by the ethics committee of university of social welfare and rehabilitation , tehran , iran . the interviewees then signed an informed consent form or verbally consented to participate in the study . table 1 compares background characteristics ( sex , education level , marital status and referral times ) between experiment and control groups . as table shows , regarding educational level , close to two thirds of experiment group belong to illiterate and primary school compare to about half percent in control group . in total , comparison of background characteristics among patients referring to ed in shahid rajaee hospital in karaj , iran in both experiment and control groups in 2011 table 2 presents time variable effect between wait time to receive ed physician first visit in experiment , and control groups . there was a significant difference between mean wait time in experiment and control groups ( 10.69 min in control vs. 8.91 min in experiment group ) . comparison of mean , median , minimum and maximum of wait time in patients referring to ed in shahid rajaee hospital in karaj , iran in both experiment and control groups in 2011 table 3 presents satisfaction in experiment and control groups that reveals higher satisfaction in triaged patients compared to control group ( p = 0.01 ) comparison of mean satisfaction in patients referring to ed in shahid rajaee hospital in karaj , iran in both experiment and control groups in 2011 table 2 presents time variable effect between wait time to receive ed physician first visit in experiment , and control groups . there was a significant difference between mean wait time in experiment and control groups ( 10.69 min in control vs. 8.91 min in experiment group ) . comparison of mean , median , minimum and maximum of wait time in patients referring to ed in shahid rajaee hospital in karaj , iran in both experiment and control groups in 2011 table 3 presents satisfaction in experiment and control groups that reveals higher satisfaction in triaged patients compared to control group ( p = 0.01 ) comparison of mean satisfaction in patients referring to ed in shahid rajaee hospital in karaj , iran in both experiment and control groups in 2011 this quasi- experimental study showed that there was a significant difference in wait time and patients satisfaction in experiment , triaged clients ; and control groups , those who received routine services . miro et al . also managed to decrease wait time through triage . as a result wait time for visiting of the patients decreased from 6.8 min to 4.5 min ( p = 0.004 ) , which is consistent with our findings in this study . however , the earlier study did not focus on patient satisfaction , as a result of the triage . moreover , tamburlini et al . with regard to evaluation of triage function in ed observed that both wait time and patients crowding could decreased after educating the nurses and establishment of a triage system in ed . this may also imply the value of nurse skills as a means of their continuous education related to their jobs , an important factor that was already pronounced in reducing pre - hospital time interval of post crash events . our study revealed that triage could significantly resulted shorten wait time and increase patients satisfaction in experiment group . in a study conducted on patients satisfaction from ed services , wait time has been indicated as an important factor by the patients and their accompanying persons . in overall hossoein zadeh , amer , anderson , edvin and trial all yielded similar results and claimed that wait time has an invert association with patients satisfaction in eds . in a study conducted in saudi arabia , close to two third of the subjects indicated wait time was the main reason for patients dissatisfaction from ed services , and observed a significant association between patients satisfaction and wait time . this also implies more care about the wait time for both emergency services and patients satisfaction . researcher presence may affect personnel 's function and possibly speed up of personnel , as a means of hawthorne effect ; however , in order to deal with it , they were confirmed that the results will keep confidentially with no report to hospital authorities . moreover , the observations of the first 3 days were left out in the data in order to diminish bias . although , hawthorne effect might have been some effect , we believed that the effect seems to be so little . in addition , due to a lack of enough alertness of 6 patients , principal investigator has to ask the questions with help of patient accompanies . however , it was just a few cases that could not affect the result of the study and its generalization to the patient as respondent . in conclusion , triage could not only significantly decrease the time interval between patients arrival and their first visit by a physician but increase patients satisfaction . with regard to the vital role of triage education in ed of hospitals and the fact that it can increase the speed of ed services , triage can improve the quantity of treatment services , as well as patients satisfaction . since , reduction of wait time interval and patient satisfaction is one important goal for nurses in emergency ward , finding from this study may improve nursing performance in relation to their work in the ed . researcher presence may affect personnel 's function and possibly speed up of personnel , as a means of hawthorne effect ; however , in order to deal with it , they were confirmed that the results will keep confidentially with no report to hospital authorities . moreover , the observations of the first 3 days were left out in the data in order to diminish bias . although , hawthorne effect might have been some effect , we believed that the effect seems to be so little . in addition , due to a lack of enough alertness of 6 patients , principal investigator has to ask the questions with help of patient accompanies . however , it was just a few cases that could not affect the result of the study and its generalization to the patient as respondent . in conclusion , triage could not only significantly decrease the time interval between patients arrival and their first visit by a physician but increase patients satisfaction . with regard to the vital role of triage education in ed of hospitals and the fact that it can increase the speed of ed services , triage can improve the quantity of treatment services , as well as patients satisfaction . since , reduction of wait time interval and patient satisfaction is one important goal for nurses in emergency ward , finding from this study may improve nursing performance in relation to their work in the ed .

accurately calculating a weak acid s pka from simulations remains a challenging task . we report a multiscale theoretical approach to calculate the free energy profile for acid ionization , resulting in accurate absolute pka values in addition to insights into the underlying mechanism . importantly , our approach minimizes empiricism by mapping electronic structure data ( qm / mm forces ) into a reactive molecular dynamics model capable of extensive sampling . consequently , the bulk property of interest ( the absolute pka ) is the natural consequence of the model , not a parameter used to fit it . this approach is applied to create reactive models of aspartic and glutamic acids . we show that these models predict the correct pka values and provide ample statistics to probe the molecular mechanism of dissociation . this analysis shows changes in the solvation structure and zundel - dominated transitions between the protonated acid , contact ion pair , and bulk solvated excess proton .

weak acid ionization plays a central role in many chemical , biological , and industrial processes . measuring and predicting the free energy of this process ( i.e. , pka values ) have become important experimental and theoretical tools , enabling control over and insight into acid / base chemistry and ph - dependent biomolecular transformations . although a large number of theoretical methods have been developed to predict pka values ( see recent reviews ( 1 ) and ( 2 ) and references therein ) , less attention has been given to understanding the molecular - scale mechanism of acid dissociation . this is largely due to the complexity and cost of analyzing the full free energy surface , which involves not only a chemical reaction but also significant solvent and solute reorganization . instead , most pka methods rely on the use of thermodynamic cycles that break the complex process of deprotonation into computationally tractable steps . for small molecules , this cycle typically combines the gas phase dissociation energy , calculated with high - level quantum mechanical ( qm ) calculations , with de / solvation free energies of the reactant and product species , calculated with continuum solvent approaches . while the gas phase energies are quite accurate , the simple continuum description of solvation free energies often requires parameter adjustment to reproduce all of the entropic and enthalpic complexities that are inherent in molecular solvation . they have been improved by hybrid approaches that include some number of explicit water molecules in the solvation free energy analysis . in conjunction with methodological refinements , these qm - continuum solvent models have enabled pka calculations of small acids to within a few units of the experimentally determined values . in contrast , the suite of methods that have been developed to predict biological pka values have typically used a thermodynamic cycle in which the solute remains in a solvated environment . each has advantages and drawbacks as well , discussed in ref ( 2 ) , however none has yet demonstrated the ability to predict pka values within a few pka units of the experimental value for all test cases . it has been suggested that challenging moieties ( e.g. , buried residues that have large pka shifts or those with strong coupling to conformational changes or other titration sites ) will require methods that better capture the underlying physics . these conclusions highlight how interesting and nuanced a biomolecular system can be . while it is clear that protonation states influence everything from stability to binding and reactivity , the details of their influence is still sometimes poorly understood . few methods to date have simulated the actual process of interest , the exchange of an excess positive charge from an acid to bulk water . to do so involves calculating the free energy surface for proton dissociation in the bulk phase environment , from which one learns about the molecular mechanism and rate . this requires three things : ( 1 ) a method that describes the underlying physics of bond formation and cleavage in the bulk phase environment , ( 2 ) a method that is efficient enough that when combined with enhanced sampling approaches can capture the distribution of phase space that defines a free energy in the condensed phase ( where entropy and energy are often a similar magnitude ) , and ( 3 ) a reaction coordinate that is flexible enough to track the transferring excess charge defect . the first of these requirements is most obviously filled by qm approaches , which have been used in a handful of helpful studies to probe acid dissociation . parrinello molecular dynamics ( cpmd ) to calculate the free energy profile of histidine dissociation . although the chosen reaction coordinate limited their analysis to stopping just beyond the transition state , they were able to calculate the correct relative pka value by subtracting the equivalent profile for water autodissociation . similarly , park et al . used cpmd in combination with metadynamics to sample the configurational space of acetic acid in bulk water . while their trajectories were too short to sufficiently converge the free energy surface , they were able to observe and characterize multiple protonation and deprotonation events . these reactions occurred along a well - characterized pathway where the excess proton is briefly shared between the acid and solvating water . they also noted the existence of a shallow minimum in the free energy surface corresponding to the existence of a contact ion pair ( cip ) that is separated from the protonated state by a small energy barrier . in an alternative approach that still describes the bond dissociation at the ab initio level , uddin et al . recently used qm / mm to calculate the free energy surfaces for several small molecules . although they obtained quite accurate pka values , their method can not describe the mechanism of dissociation since they include only one water molecule and the acid in the qm region . hence , this method changes the physical process from the dissociation of a delocalized charge defect to proton transfer followed by separation of a localized hydronium cation and acid anion in a solvating mm environment . just as for thermodynamic cycles , their results show that sampling an alternative pathway between reactant and product states can yield accurate pka values for small molecules . another approach that describes the bond dissociation process is reactive md ( rmd ) . the efficiency of these models allows them to sample reactive dynamics on much longer length and time scales than ab initio md . since they are parametrized to work with mm force fields , they also avoid the boundary issues suffered by qm / mm calculations . both of these factors make rmd models ideal for simulating reactions in large , complex systems with slow dynamics . proteins and proton exchange membranes are two important examples of such systems . without sufficient sampling in these types of systems , the final result can be strongly influenced by the initial conditions . a common criticism of rmd models is that they are only approximations to the true quantum mechanical behavior of the system . while this is certainly true , if the functional form is both flexible enough and parametrized correctly , then the reactive model will retain the ability to accurately reproduce the potential and free energy surfaces from which it was parametrized . the multistate empirical valence bond ( ms - evb ) method is a rmd force field that has been used to characterize proton transport in many condensed phase and biological environments . it is especially well suited to the challenge of acid ionization since it inherently defines a center of excess charge that can be used to track reactions involving proton transport , thereby avoiding issues with geometric reaction coordinates . although ms - evb has been used to study weak acids and amino acid dissociation , these efforts involved a larger degree of empiricism since the pka value was needed to fit the msevb potential . it has been a long sought goal to find a procedure that would minimize the empiricism in rmd models , such that the bulk property of interest was derived entirely from quantum data in combination with extensive sampling . this work reports the achievement of that goal , a multiscale approach to calculate the free energy profile for acid ionization that is based entirely on qm data and yields an accurate absolute pka value . an iterative procedure , in the spirit of adaptive force matching , is used to sample the reactive phase space , obtain reference ab initio data ( qm / mm forces ) , and fit a rmd potential . care has been taken to modify the functional form such that it is sufficiently flexible to reproduce the qm / mm reference data and , in principle , capture the essential physics . our approach is applied to aspartic acid ( asp ) and to glutamic acid ( glu ) . both of these amino acids play crucial roles in proton and ion transport in biological channels and pumps and are also commonly involved in salt bridges that influence protein dynamics , structure , and binding . we show that accurate estimates of the pka values of both asp and glu are obtained from the resulting reactive models , which is the first time a reactive model has been able to accurately predict the correct pka without being explicitly parametrized to do so . finally , we discuss the mechanistic description our models provide of acid deprotonation in water . to emphasize that these models were fit entirely to qm / mm data and without adjustments to match the experimental pka , we will hereafter refer to them as multiscale reactive molecular dynamics ( ms - rmd ) models . it should be noted , however , that the ms - rmd asp and glu models are parametrized to work with a refined version of the ms - evb3 hydronium model that accurately describes a proton solvation and transport in water . ms - rmd is similar to the ms - evb framework that has been successfully used to model proton solvation and transport in many aqueous systems . the power in this approach lies in its ability to describe electronic delocalization as a linear combination of topologically distinct states . for a given configuration , for example , state |i could be a protonated amino acid in a box of water ( figure 1a ) . state |j would have the same coordinates but have a topology describing a cip between a solvated hydronium ion and a deprotonated amino acid ( figure 1b ) . at each step in the simulation , a state search algorithm first determines all the possible states based on geometric criteria and then calculates the energy of each state . the lowest energy state is termed the pivot state and is the start of the state search algorithm at the next step . since the number of waters , and therefore the number of possible states , grows exponentially with distance from the pivot state , only states in the first three solvation shells several different bonding topologies are shown for a given set of nuclear coordinates , r. dashed lines surround the protonated molecule , as defined by the bonding topology . the reactive system is described by the following hamiltonian:1where r is the vector describing the nuclear coordinates , hij is the sum of mm potential terms for state i , and hij is the coupling between states |i and |j. the details of these terms will be provided in the force field section . with the hamiltonian defined this way , the relative weight of each state is described by the components of the eigenvector , ci:2finally , forces are calculated using the hellmann feynman theorem . these forces are then passed to a standard md integrator such as velocity verlet . free energy calculations require a continuously varying reaction coordinate ( rc ) to describe the process of interest . the evb formalism provides a convenient reference for the rc ; the center of the excess charge ( cec):3 in this equation , ri is the geometric center - of - charge for the protonated molecule in state i , and ci is the relative weight of that state . we then use the distance between the cec and the center of mass of the asp or glu carboxyl group to describe the progress of the deprotonation reaction . the diagonal elements of the ms - rmd hamiltonian matrix are derived from the standard charmm mm force field . the protonated form uses all of the standard parameters with the exception of the acidic o h bond . this bond is replaced with a morse potential to allow dissociation:4where r is the bond length and d , , and r0 are parameters fit to reproduce the bond length and frequency of the harmonic potential it replaces while still allowing for a reasonable bond dissociation energy . the deprotonated form of amino acid uses all of the standard charmm bonded and nonbonded terms and parameters . since the charmm force field was not originally intended for use in reactive simulations , additional repulsive terms are used between the carboxyl oxygens of the deprotonated model and the hydronium oxygen and hydrogens . these terms help to correct for the overattraction at close distances due to the use of point charges . the functional forms for the repulsions are the same as those used in the ms - evb3 hydronium model:56where b , b , b , c , and c are fitted parameters . following the logic in previous work , and to avoid parameter redundancy , doo and doh were fixed the same values used in ms - evb3 . the sum of gaussians term in eq 5 is included to help the fitted potential to reproduce the correct asymmetric solvation structure around the hydronium , and the same switching function is used as given in eq 9 of ref ( 28 ) . the asp and glu models are designed to work with a refined version of the ms - evb3 force field , herein referred to as msevb 3.1 . this refinement uses the same functional forms for the hamiltonian but revised parameters . using a genetic algorithm , the fitting parameters were optimized using the original ab initio data sets as well as potential energy surface scans for the eigen ion at the same level of the original ab initio calculations . furthermore , the oxygen partial charge of the hydronium is an adjustable parameter ( the charge for the hydrogens is automatically determined to maintain the hydronium + 1 total charge ) . parameters for this force field are presented in the supporting information . in this study , the limiting species are the de / protonated asp or glu models and the ms - evb 3.1 solvated proton in spc - fw water . the ms - rmd force field is parametrized to smoothly switch from one protonated species to another . the asymmetry of the reaction necessitates several modifications to the original ms - evb equations , which were designed with water in mind . the off diagonal coupling term , hij , is designed to reproduce the correct transition state geometry . previous ms - evb models used a complex off - diagonal term to describe a symmetric reaction pathway between two waters . however , the asymmetry of the asp - water system was better fit by a simple gaussian potential:7where roh is the distance between the deprotonated asp oxygen and the excess proton . the other parameters determine the shape and size of the gaussian and are fit to the qm / mm data . in practice , protonation reactions occurring between different species require a constant energy term ( vii ) to be added to the deprotonated state . this term accounts for the difference in energy resulting from different mm force fields . without it , the protonated and deprotonated asp potential energy surfaces would be offset by hundreds of kilocalories per mole , and no reaction would ever take place . this difference is due primarily to electrostatics and can be estimated by subtracting the coulomb energy of the most favorable hydronium state ( containing the favorable electrostatic interactions between the hydronium cation and amino acid anion ) from the coulomb energy of the protonated ( and neutral ) amino acid state . figure 2 shows a plot of the average value of this term as a function of rc . since vii controls the relative stability of the protonated and deprotonated states , it was used in past work to tune the behavior of the model to reproduce experimental quantities such as pka . in the current work , an iterative fitting approach is used to rigorously determine the value of vii along with all other ms - rmd parameters . plot of the difference in coulomb energy between the protonated asp state and the lowest energy non - asp state . at short distances , it is similar in some ways to that used in previous work in which ms - rmd models were force matched from aimd data . there are , however , a few important differences , including the use of qm / mm instead of aimd , the use of empirical functional forms instead of tabulated potentials , and the use of an iterative scheme for sampling configuration space starting from a guessed hamiltonian . these choices were partially motivated by the success that has been demonstrated with the adaptive force matching method by wang and co - workers . reactive hamiltonian in order to generate a set of configurations along the reactive pathway . next , high - level qm / mm calculations are performed to collect the atomic forces for those configurations . the new reactive hamiltonian is then used to generate new configurations via umbrella sampling , and the process is repeated until the parameters reach the desired convergence . details of each step are discussed in the text . due to the nonlinearity of several of the reactive force field parameters , the reactive model is fit to the qm / mm reference data using a genetic algorithm to minimize the residual:8here , nc and na are the number of configurations and the number of atoms in each configuration , respectively . w(rij ) is the weight for each atom that , unless specified otherwise , is set to 1 . fij is the atomic force from the current ms - rmd parameter set and fijqm / mm is the reference force from the qm / mm calculation . the fitting calculations were run in parallel and used 4000 genomes ( parameter sets ) per generation . to lessen the danger of overfitting the first step followed the uniformly distributed mutation scheme outlined in section 3.1 of ref ( 40 ) . the range of parameters was chosen to be as broad as possible without allowing unreasonable values . for example , c3 in the off - diagonal term would be unreasonable if it were greater than the aspo once a converged parameter set was reached with the uniform distribution , the mutation scheme was switched to the normally distributed scheme described in section 3.2 of ref ( 40 ) . this scheme is very good at quickly finding a local minimum and was therefore used to find the best genome in the vicinity of the result from the first step . since the second scheme is easily trapped in local minima , it was not ideal for use on its own . the combination described here was found to quickly and reliably converge on the optimum solution . the discrete recombination scheme described in section 2.2 of ref ( 40 ) is used with both mutation schemes . the nature of the reactive model introduces additional complexity into this otherwise simple scheme . vii , which directly controls the depth of the protonated well relative to the deprotonated cip , is a constant that is only included for states in which the amino acid is protonated . the off - diagonal term acts over a broader range spanning the protonated amino acid and cip configurations but also has a negligible effect once the cip dissociates . in contrast , the repulsive terms , which are only applied to the deprotonated amino acid , are significant at longer distances and negligible at distances below the transition state . furthermore , since both vii and the repulsive terms are added to the diagonal matrix elements , they tend to have correlated behavior if fit simultaneously . thus , the value of vii for the first iteration after generating new qm / mm data is chosen to be the average value described in figure 2 . this value has been found to be within a few kilocalories per mole of the final value of vii and is therefore a good initial approximation . holding the value of vii fixed , the off - diagonal and repulsive terms can be fit to the full set of qm / mm reference forces . once the off - diagonal and repulsive terms reach their optimum value for the current value of vii , umbrella sampling is used to generate a pmf with the model . since vii s effect is most important at and around the transition state , a range of rc values immediately surrounding the transition state is chosen from the pmf , and configurations from that range are then used to fit a new value of vii . using this new value of vii , the off - diagonal and repulsive terms are refit while vii is again held constant . this procedure fits an empirical model from solely qm / mm data and results in models that correctly predict the bulk phase pka values for both asp and glu . solution simulations of asp and glu include one solute molecule solvated by 486 and 467 waters , respectively . to reduce interactions with the charged backbone protonatable sites , the n - termini were capped with an acetyl group and the c - termini were capped with methylamine . the systems were equilibrated in the npt ensemble at 1 atm and 310 k for 200 ps before changing to the nvt ensemble with a cubic box of 24.55486 on a side for asp and 24.24938 for glu . all reactive simulations were run with a modified version of the lammps simulation package . the rc was chosen to be the distance between the cec and the center - of - mass of the amino acid s side chain carboxylic acid . umbrella sampling was used to evenly sample the rc . for the asp system , 25 windows were spaced every 0.36 starting at a rc value of 1.0 . the glu system used 36 windows spaced every 0.25 with 20.0 kcal / mol restraints . each production window was further equilibrated for 100 ps , and then statistics were collected for 1.7 ns . the pka is calculated from the resulting pmf using the following equation:9where w(r ) is the free energy from the pmf and is the product of the simulation temperature and the boltzmann constant . the integral is calculated from zero to the transition state , as denoted by . c is the standard state concentration whose value is 1660 / molecule and results from the entropic freedom that is gained by the proton when it dissociates from the acid . atomistic configurations for the qm / mm calculations were selected from the umbrella sampling trajectories . in order to ensure a uniform distribution along the rc , individual configurations were sorted by their rc value into 60 0.067 wide bins . five configurations were then selected at random from each bin , giving a total of 300 configurations for each iteration . qm / mm was performed in cp2k using b3lyp with the double - zeta basis set . unrestricted dft was used to allow the most accurate calculation of forces near the dissociation barrier . b3lyp was chosen over mp2 because recent studies have shown comparable accuracy with a reduced computational cost for water and the solvated excess proton . calculations were run on 256 intel sandy bridge cores and on average completed within 10 min . the qm region included the entire amino acid residue and all of the waters found by the ms - rmd state search . because proximity to classical point charges reduces the accuracy of forces calculated on qm atoms , an additional 3 buffer layer of qm water was added around the qm atoms . all together , this results in a qm region containing an average of 208 atoms , or one asp / glu and 61 waters . in total , four iterations were needed for asp parameters to converge , while glu parameters converged in three iterations . parameters for the new asp and glu models are presented in table 1 . these parameters were fit to qm / mm reference forces using the previously described iterative scheme to ensure convergence . it must be noted that the asp and glu models were parametrized using the same method , but entirely independent data sets . the fact that the final parameters for each model are so similar is not surprising , however , given the molecular similarity and small difference in experimental pka values . as the next section discusses , the pka predicted by each model is a very good estimate of the experimental value . the fact that the models are able to capture such subtle differences is evidence that the methodology works well . the potential of mean force ( pmf ) of deprotonation describes the free energy of the process as a function of the rc . to obtain a free energy change between any two points on the pmf , one simply integrates the pmf . figure 4a shows the pmf for deprotonation of asp , and figure 4b shows the pmf for glu ( black lines ) . as depicted in figure 5 , the rc gives the distance between the center - of - mass of the carboxylic moiety and the cec . rc values around 1.3 correspond to the protonated amino acid , while values between 2.1 and 5 correspond to the stable cip . values above 5.5 represent an excess proton separated from the anionic acid by bulk solvent . pmfs and cmax distributions from the final ( a ) asp and ( b ) glu models . the colormap in the background gives the cmax probability distribution normalized for each rc value . this distribution describes the extent of charge delocalization and , by extension , the solvation structure . snapshots from umbrella sampling trajectories illustrating the types of structures suggested by the cmax distributions and the 2d - rdfs . arrows indicate the region of the pmf where the windows were run , while the distances in the box are the center of the harmonic restraint . the position of the cec is shown as a yellow sphere . in eigen structures , the cec is nearly aligned with the central water s oxygen . in zundel structures , the protonated forms of asp and glu are much more stable than the deprotonated forms , which is consistent with their status as weak acids . evaluating the integral in eq 9 estimates the pka values for asp and glu to be 3.82 0.17 and 4.56 0.18 , respectively . these are both in very good agreement with the experimental ranges of 3.71 to 3.90 for asp and 4.15 to 4.31 for glu . the barrier that prevents protonation is due to the cost of forming a zundel - like arrangement between the acid and hydronium . the depth and extent of the cip varies with the system ; however its existence has been predicted in a variety of weak acid systems simulated with different methods . when preparing a simulation to calculate a pmf of deprotonation and a pka , care must be taken to ensure that the rc is sampled far enough beyond the cip to ensure bulk - like properties . if this is not done , then the pmf from which the pka is calculated will be shifted and the resulting pka will neither be accurate nor reflect the quality of the model . fortunately , sampling well beyond the cip is not a problem with ms - rmd models due to their efficiency . however , care needs to be taken when running qm / mm simulations to ensure that a large enough qm box and enough solvating waters are used to not only sample beyond the cip but also to avoid boundary issues . with the pmf in hand , one the ms - rmd methodology conveniently provides a measure of the structure of the solvated complex . it has been shown that the square of the largest component of the ground state eigenvector ( cmax , from c in eq 3 ) describes the solvated structure . a value of 1 indicates the limiting case of an entirely localized excess charge , while values less than that quantify the degree of charge delocalization . eigen structures , where the excess charge is shared among a central hydronium and three h - bound water molecules , have a cmax value around 0.65 . zundel structures , which share the excess charge more equally between two water molecules , have a cmax value closer to 0.5 . it should be emphasized that there is always additional delocalization to other surrounding water molecules , even for these limiting eigen- and zundel - like cations . by plotting the cmax probability density as a function of the rc , we gain insight into the structural changes that accompany deprotonation . this probability density is plotted as a colormap in figure 4 . at low rc values , both asp and glu have an average cmax greater than 0.9 , which indicates that the structure and dynamics mostly resemble the limiting case of a protonated acid in bulk water with little charge transfer to the surrounding water molecules . this is notably different than the protonated water molecule , which always involves a significant amount of charge delocalization . as the proton begins to dissociate , the cmax decreases as the proton goes through a clear zundel - like transition between the acid and solvating water . beyond the transition state , the system begins to resemble an eigen cation where one of the solvating waters is replaced with the acid anion . however , the eigen structure seen in the cip is distinctly different from that seen in bulk water , as evidenced by a cmax of 0.73 . the close proximity of the anionic acid helps to stabilize the hydronium , leading to a more localized charge . further evidence of the uniqueness of the cip is that there is a well - defined zundel transition around 4 linking the cip to the bulk - like structure at longer distances . eigen transitions in bulk , the water around the anionic amino acid is more structured , as evident in the distinct zundel - like transition at a rc value around 4 . figure 5 shows snapshots of the typical structures found at different rc values and characteristic of significant regions in the pmf . the bonding topology reflects o h distances less than or equal to 1.6 , which is commonly considered to be the length of a hydrogen bond . it is also interesting to look at the structural changes that occur directly around the cec . to do so , the two - dimensional radial distribution functions ( 2d - rdfs ) between the cec and surrounding water oxygens ( ow ) and water hydrogens ( hw ) are shown in figure 6 . a slice along the first dimension of the 2d - rdfs gives a traditional 1d - rdf , which describes the probability of finding the indicated pair a given distance apart . scanning the second dimension shows how the solvation structure changes as the reaction progresses according to the rc . in combination with the cmax analysis , the 2d - rdfs show that the solvent has well - defined rearrangements as the proton dissociates from asp and travels to bulk . figure 6a shows the 2d - rdf for asp s cec ow distance . once asp is deprotonated , the first peak is near 2.5 , which is characteristic of the eigen structure . when the rc reaches 4 , the peak distance drops to around 1.5 , which is characteristic of a slightly asymmetric zundel cation where the cec resides close to the midpoint of the o o vector . the 2d - rdf showing asp s cec 2d - rdfs for ( a ) cec ow and ( b ) cec hw with asp . these plots show how the water structure changes around the cec as deprotonation occurs . 2d - rdfs for ( a ) cec ow and ( b ) cec hw with glu . we have presented a procedure for fitting the parameters of a ms - rmd model to reproduce the forces from qm / mm calculations . due to the efficiency of ms - rmd , we are able to collect and analyze data from large time and length scales that can reveal intricate behavior and is essential for converged bulk phase thermodynamic properties . this new methodology was used to parametrize new models of both asp and glu that correctly predict the correct experimental pka values . this is the first time that a ms - rmd model has been able to reproduce the experimental pka values without any fitting to experimental data . multi - nanosecond trajectories were analyzed to extract important information about the mechanism underlying the deprotonation process . it is shown that the deprotonation process follows a well - defined series of zundel to eigen transitions as the excess proton travels from asp to bulk solution . this work is considered continued progress toward the goal of having flexible and systematic algorithms to reliably make a multiscale connection between accurate electronic structure calculations and efficient empirical models to describe complex reactive processes . as more accurate ab initio methodologies ( e.g. , those explicitly accounting for electronic correlation ) become computationally tractable for qm / mm of condensed phase systems , the procedure presented herein and related force matching algorithms will be invaluable tools for mapping high level data into efficient rmd models that can significantly extend the time and length scales of reactive simulations to explore complex phenomena . while the results presented herein are important , the true test of this procedure will be to apply it in biological systems where the pka of individual residues can be shifted by several pka units . proton transport in proteins is very sensitive to the underlying protonatable residue models , and it is therefore crucial to have a procedure to ensure that those models are physically accurate . furthermore , biological systems are orders of magnitude larger and have much slower dynamics than the systems presented here . while it is conceivable that qm / mm md simulations could reach the same sort of time scales as the solution simulations presented here , they would be prohibitively expensive to run the length - and time - scales needed to account for the slow dynamics commonly found in biological processes . the ms - rmd methodology has already been shown to scale well to hundreds of thousands of atoms and can realistically reach tens of nanosecond simulations with large explicit biomembrane systems . work is already underway to implement these reactive models in protein environments and to use the presented methodology to fine - tune these and other individual amino acid models to behave properly in their specific protein environments .

new treatments , such as sipuleucel - t and androgen receptor- ( ar- ) directed therapies ( enzalutamide ( enz ) and abiraterone acetate ( aa ) ) , have emerged and been approved for the management of castration - resistant prostate cancer ( crpc ) . there are still debates over their efficacy and clinical benefits . this meta - analysis aimed to investigate the efficacy and safety of sipuleucel - t and ar - directed therapies in patients with crpc . revman 5.1 was used for pooled analysis and analysis of publication bias . seven studies were included in the meta - analysis , with three studies in sipuleucel - t ( totally 737 patients , 488 patients in treatment group , and 249 patients in placebo group ) and four in ar - directed therapies ( totally 5,199 patients , 3,015 patients in treatment group , and 2,184 patients in placebo group ) . treatment with sipuleucel - t significantly improved overall survival in patients with crpc and was not associated with increased risk of adverse event of grade 3 ( p > 0.05 ) . however , treatment with sipuleucel - t did not improve time - to - progression and reduction of prostate - specific antigen ( psa ) level 50% was not significantly different from that with placebo . ar - directed therapies significantly improved overall survival in patients with crpc and improved time - to - progression and reduction of psa level 50% . ar - directed therapies did not increase risk of adverse event of grade 3 ( p > 0.05 ) .

worldwide , in 2015 , it is the second most common newly diagnosed cancer and the fourth most common cause of cancer death in men . in the united states , incidence and mortality of prostate cancer ranked first and second , respectively , in men . over the past few years , incidence of prostate cancer increased steadily , with slowly increased mortality [ 13 ] . current treatments for prostate cancer include surgical and medically induced castration and androgen deprivation therapy ( adt ) using androgen receptor ( ar ) antagonists . despite these treatments , castrate - resistant prostate cancer ( crpc ) is defined as disease progression despite adt and may present a spectrum of disease ranging from rising prostate - specific antigen ( psa ) levels , progression of preexisting disease , or appearance of new metastases [ 68 ] . docetaxel , approved by the us food and drug administration ( fda ) in 2004 , is a taxane drug that induces polymerization of microtubules and phosphorylation of bcl-2 protein . three weeks of combined docetaxel and prednisone is currently considered the standard of first - line chemotherapy for men with crpc . the second - line chemotherapy with cabazitaxel has been shown to increased survival time in patients with crpc . however , severe adverse events have been reported for these treatments [ 10 , 11 ] . with advances in the understanding of disease pathophysiology , new treatments for crpc emerge in the recent years that aim to improve both survival and quality - of - life of patients . these treatments include cancer immunotherapy such as sipuleucel - t , ar - directed therapies such as abiraterone acetate ( aa ) and enzalutamide ( enz ) , radium-223 , and prostvac [ 1318 ] . phase iii clinical trials have been conducted for sipuleucel - t , aa , and enz and fda has approved their use in patients with crpc [ 2123 ] . these new treatments hold great potential as the first - line treatments for patients with crpc . finding the optimal regimen is now the major clinical challenge . this meta - analysis aimed to investigate and compare the efficacy and safety of these two treatments and to provide scientific evidence for the management of crpc . aa is a steroidal antiandrogen that exerts its effect through inhibiting cyp17a and it also acts as an antagonist of ar [ 24 , 25 ] . it has a strong binding affinity for ar and in addition prevents binding of ar to deoxyribonucleic acid and ar to coactivator proteins . the antigen presenting cells ( apcs ) are harvested from individual patient 's peripheral blood and later incubated with recombinant fusion protein antigen , which contains both prostatic acid phosphatase and granulocyte - macrophage colony - stimulating factor [ 27 , 28 ] . this process activates the apcs , which are critical for priming a cytotoxic t - lymphocyte - mediated immune response . these activated apcs are then reinfused into the individual patient . in 2010 , sipuleucel - t became the first immune - therapeutic agent approved by the fda for patients with crpc , based on consistent observed improvement in overall survival . this meta - analysis aimed to further determine the clinical efficacy and safety of these two types of treatments , namely , sipuleucel - t and ar - directed therapies ( aa and enz ) , in the management of crpc . survival and disease progression were assessed by overall survival ( os ) and time - to - progression ( ttp ) , respectively . we systematically searched seven literature databases ( ovid , springer , pubmed , web of science , sciencedirect , medline , and cochrane library ) from 1966 to october 2015 for all relevant articles by entering terms including castrate - resistant prostate cancer , sipuleucel - t , enzalutamide , and abiraterone acetate as key words , title , subject heading , and text word . we also searched for potentially missed articles from the reference list of retrieved articles and from previous narrative reviews on this topic . studies were included if they met the following criteria : ( 1 ) randomized double - blind place - controlled clinical trials of sipuleucel - t , aa , and enz presenting original data ; ( 2 ) patients with crpc ; ( 3 ) english articles published before october 2015 . in case of duplicated reports , the article presenting the latest and the most comprehensive data on the largest cohorts was selected . studies were excluded if ( 1 ) they were duplicated reports , were of poor quality , were lacking original data , or presented incomplete data ; ( 2 ) they were review articles , conference abstracts , or commentary . two authors ( renliang yi and baoxin chen ) conducted literature search and study selection independently . results were compared and discrepancies were resolved by a discussion with another author ( peng duan ) . quality of the included articles was assessed using the newcastle - ottawa scale ( nos ) , in which a study is judged on three broad perspectives : the selection of the study groups ( adequate definition of the cases , representativeness of the cases , selection of controls , and definition of controls ) , the comparability of the groups ( compatibility of cases and controls ) , and the ascertainment of the outcome of interest ( ascertainment of exposure , ascertainment of cases and controls , and nonresponse rate ) . results were compared and discrepancies were resolved by a discussion with another author ( weilin ou ) . the following outcomes were extracted from each study : os , ttp , reduction of psa level 50% , and adverse events of grade 3 . results were compared and discrepancies were resolved by a discussion with corresponding author ( zhiheng zhou ) . review manager 5.1 software was used for data synthesis and analysis . the hazard ratio ( hr ) with 95% confidence interval ( ci ) heterogeneity analysis was performed using q test with p > 0.1 and i < 50% suggesting homogeneity among studies . for data without significant heterogeneity , fixed - effect models were used for pooled analyses . in case of significant heterogeneity , sensitivity analysis was performed by excluding the study with the highest variance . in the case that no definite cause was found for heterogeneity , random - effect model was used for pooled analyses . the significance of pooled data was further tested and a p < 0.05 was considered statistically significant . when enough studies were included , funnel plot was delineated and the publication bias was evaluated . a total of 302 articles were excluded after reviewing titles and abstracts , and 80 articles were excluded due to duplicated reports . exclusion reasons included review articles and commentary , correspondence , nonrandomized placebo - controlled trials , and lack of complete study outcomes . seven articles were included for the meta - analysis : three articles on sipuleucel - t , two on aa , and two on enz , respectively . all seven studies were randomized , double - blind , placebo - controlled clinical trials . results of quality assessment using nos for the seven studies are showed in table 1 . regimens used in these studies were as follows : ( 1 ) sipuleucel - t : patients were randomly assigned in a 2 : 1 ratio to receive either sipuleucel - t or placebo every two weeks , for a total of three infusions ; ( 2 ) aa : intervention group received combined aa 1000 mg and prednisone 10 mg daily and placebo group received prednisone 10 mg daily plus placebo ; ( 3 ) enz : intervention group received enz 60 mg daily and placebo group received placebo . all seven studies provided data on survival with follow - up period up to 36 months [ 29 , 3136 ] . analyses of os were performed in 5,936 patients , with 737 patients for sipuleucel - t ( intervention group versus placebo group : 488 versus 249 patients ) and 5,199 patients for ar - directed therapies ( intervention group versus placebo group : 3,015 versus 2,184 patients ) . results showed that , compared with placebo , both sipuleucel - t and ar - directed therapies significantly improved survival of patient with crpc . pooled hr for os was 0.73 for sipuleucel - t ( 95% ci : 0.610.88 ; z = 3.31 ; p < 0.001 ) and 0.72 for ar - directed therapies ( 95% ci : 0.660.78 ; z = 7.94 ; p < 0.00001 ) . tests for heterogeneity showed insignificant results , indicating homogeneity among studies ( both p > 0.1 and both i < 50% ) . six studies with a total of 5,936 patients reported ttp [ 29 , 3135 ] , including 737 patients for sipuleucel - t ( intervention group versus placebo group : 488 versus 249 patients ) and 5,199 patients for ar - directed therapies ( intervention group versus placebo group : 3,015 versus 2,184 patients ) . compared with placebo , sipuleucel - t showed no significant favorable effect on ttp with pooled hr of 0.88 ( 95% ci : 0.741.06 ; z = 1.35 ; p = 0.18 ) . test for heterogeneity was not significant ( p = 0.35 , i = 4% ) . in contrast , ar - directed therapies showed significant improvement in ttp with pooled hr of 0.59 ( 95% ci : 0.400.88 ; z = 2.59 ; p = 0.009 ) . seven studies with a total of 5,936 patients reported reduction of psa level 50% as study outcome [ 29 , 3136 ] , including 689 patients for sipuleucel - t ( intervention group versus placebo group : 458 versus 231 patients ) and 4,975 patients for ar - directed therapies ( intervention group versus placebo group : 2,928 versus 2,047 patients ) . pooled rr showed that sipuleucel - t has no significant effect on reducing psa level 50% ( rr : 2.51 ; 95% ci : 0.659.73 ; z = 1.33 ; p = 0.18 ) . test for heterogeneity was not significant ( p = 0.5 ; i = 0% ) . in contrast , ar - direct therapies showed significant effect on reducing psa level 50% ( rr : 9.82 ; 95% ci : 1.9948.46 ; z = 2.89 ; p = 0.004 ) ( figure 4 ) . to investigate the safety of these treatments , we compared the occurrence of adverse events of grade 3 , including fatigue , headache , back pain , arthralgia , constipation , and diarrhea , with that in placebo . pooled rr revealed that , compared with placebo , risk of adverse event was not significantly increased for sipuleucel - t and ar - directed therapies ( p > 0.05 , table 3 and figure 5 ) . figure 6 shows symmetry funnel plot , indicating that there was no significant evidence of publication bias . researches on novel treatments for crpc have gained increasing interest in the past few years , especially those on sipuleucel - t and ar - directed therapies . this meta - analysis investigated the efficacy and safety of sipuleucel - t and ar - directed therapies , providing valuable information that might be useful clinical evidence on the treatments for crpc . we found that both sipuleucel - t and ar - directed therapies could significantly improve os in patients with crpc , with favorable safety . ar - directed therapies appear to have superior effects in improving ttp and in reduction of psa level . however , there are still debates over the efficacy and optimal regimen of these new treatment methods . it has been known that traditional chemotherapeutic drugs lacked the selectivity on target tumor cells , which may cause different damage to normal cells , or even serious effects on patients . indicated that adverse effects of docetaxel including edema , neurotoxicity , and hair loss limit its application . zhou et al . also showed 2.7% of crpc patients died after docetaxel plus prednisone therapy , 58.56% had neutropenia , and 19.82% had leukopenia . unlike the traditional ones , sipuleucel - t and ar - directed therapies target tumor cells , thus causing little toxic effects on normal cells due to high selectivity . for instance , aa and enz antagonize androgen receptors to inhibit the activity of tumor cells . similarly , sipuleucel - t could elicit immune response targeting against antigen prostatic acid phosphatase ( pap ) that is highly expressed in most prostate cancer cells [ 39 , 40 ] . many results showed that sipuleucel - t and ar - directed therapies improved the overall survival [ 29 , 3136 ] by exerting different effects on ttp , psa level , and aes . although surgery , radiotherapy , or chemotherapy had stopped for a period of time before the new drugs clinical trials were given , it is still hard to rule out the possibility of the influence by the former treatments . therefore , further clinical validation is needed . beyond that , we paid more attention to the sequela , applicable scopes , and contraindications of these treatments . all trials had strict selection of patients . to be an eligible case for sipuleucel - t trial , histological confirmation on castrate - resistant prostate cancer , serum testosterone level < 50 ng / dl , and a considerable somatic function for expected survival were required . patients accepted for aa trial should had no more than two previous chemotherapies , at least one previous docetaxel therapy , and mild symptoms or no symptoms ( radiographic progression in soft tissue or bone with or without psa progression , psa < 50 ng / dl , and ecog performance status of 2 or less ) . patients from sipuleucel - t trials were scheduled to undergo leukapheresis procedures every 2 week for a total of three times , and on the second day after each leukapheresis procedure , patients were treated by infusion of sipuleucel - t or placebo . patients from aa trials received abiraterone acetate 1000 mg once daily plus prednisone 5 mg twice daily by oral or placebo plus prednisone . and in enz trials patients received enzalutamide 160 mg orally once daily or matched placebo . we found that in aa trials patients with lower score of ecog , age 65 years , and psa level > or < median had the higher hr . on the contrary , in enzalutamide trials , patients with age 65 years , higher score of ecog , and psa level > median had the higher hr . while in sipuleucel - t trials , patients with age < median , psa < or > median , and higher score of ecog had the higher hr . drake 2012 indicated that the subgroup of patients aged less than 65 years did not favor sipuleucel - t . another observation by them was the potential harm from the impact study interventions , because , to some extent , sipuleucel - t broke the immune balance . challenges remain in finding the optimal regimen for sipuleucel - t and ar - directed therapies . combined sipuleucel - t , aa , and prednisone formula has been proposed as a novel treatment diagram in crpc . research has shown that concurrent administration of aa and prednisone did not blunt immunologic effects or alter immune parameters that correlate with sipuleucel - t 's clinical benefits . cumulative apc activation , cumulative apc number , total nucleated cell counts , and immune responses to sipuleucel - t were not affected by coadministration of aa and prednisone . such combination of treatments was well tolerated , with no new risk marker emerging . sipuleucel - t is recommended as the first - line treatment for patients with crpc by the national comprehensive cancer network and it is recommended as early use in asymptomatic crpc or patients with mild symptoms . this was possibly a result of mutation of ar induced by prednisone , which will subsequently impact the effect of enz on ar . therefore , it appears that there is limited clinical benefit for combination or sequential use of enz and aa [ 4548 ] .

to examine the relationship between dietary energy density ( ded ) and the nutritional quality of the diet , using data from the irish national children 's food survey ( ncfs ) and the national teens ' food survey ( ntfs ) , two cross - sectional studies of food consumption were carried out between 2003 and 2006 . data from the ncfs and ntfs were used to examine the intakes of nutrients and foods among those with low- ( ncfs < 756 , ntfs < 765 kj / g ) , medium- ( ncfs 756875 , ntfs 766885 kj / g ) and high - energy - dense diets ( ncfs > 875 , ntfs > 885 kj / g ) . a 7-d food diary was used to collect food intake data from children ( n 594 ) and teenagers ( n 441 ) . ded ( kj / g ) was calculated including food alone and excluding beverages . participants with lower ded consumed more food ( weight ) but not more energy . they also consumed less fat and added sugars and more protein , carbohydrates , starch and dietary fibre and had higher intakes of micronutrients . participants with lower ded had food intake patterns that adhered more closely to food - based dietary guidelines . low ded was associated with multiple individual indicators of a better nutritional quality of the diet , including higher intakes of dietary fibre and micronutrients and a generally better balance of macronutrients , as well as being associated with food intake patterns that were closer to healthy eating guidelines . taken together , these findings support the conclusion that a low ded may be an indicator of a better nutritional quality of the diet .

the national children 's food survey ( ncfs ) 20032004 and the national teens ' food survey ( ntfs ) 20052006 were carried out to establish databases of habitual food and drink consumption in representative samples of irish children aged 512 years and teenagers aged 1317 years . a 7-d weighed food record was used to collect food intake data from 594 children ( 293 boys , 301 girls ) , while a 7-d semi - weighed food record was used to collect food intake data from 441 teenagers ( 224 boys , 217 girls ) . participants and their parents / guardians were visited by a trained nutritionist four times , including one training visit , during the recording period . participants with the aid of their parents / guardians ( younger children requiring more aid up to 100 % ) were asked to record detailed information regarding the amount and types of all foods , beverages and supplements consumed over the 7-d period and , where applicable , the cooking method used , the brand name of the food consumed , packaging size and type and details of recipes and any leftover . a hierarchal method for dietary data collection and quantification was used which included weighing , photographic food atlases , manufacturers information and household measurements . analyses of dietary intake data were carried out using wisp ( tinuviel software ) , which contains mccance and widdowson 's the composition of foods , 6th edition and the irish food composition database . self - reported health and lifestyle questionnaires were completed by participants or their parents / guardians concurrent with collection of food intake data . this study was conducted according to the guidelines laid down in the declaration of helsinki and all procedures involving human participants were approved by the st . james ' hospital and federated dublin voluntary hospitals joint research ethics committee for the ncfs and the university college cork clinical research ethics committee of the cork teaching hospitals for the ntfs . written informed consent was given by the children , teenagers and their parents or guardians . children were selected from twenty - eight primary schools in the republic of ireland from a database obtained from the department of education and science . the database was divided into ( a ) small / medium / large schools , ( b ) all boys / all girls / mixed , ( c ) disadvantage / not disadvantaged and ( d ) urban / rural . a random sample was selected from each category so that in the final sample the proportions of children attending each of the categories of schools reflected that of the proportions according to the database . the principal at selected schools was given detailed instructions on how to select children for participation in the survey from the school roll . teenagers were recruited in a similar vein from thirty - two secondary schools with a divide of secondary / vocational / comprehension schools in place of the small / medium / large for children . the overall response rate was 66 % for children and 63 % for teenagers . analysis of the demographic features of the samples has shown them to be representative samples of irish children and teenagers with respect to age , sex , social class , socio - economic group and geographic location when compared with census data . ded ( kj / g ) was calculated including food only and excluding all beverages . this included all solid foods and liquid - like foods such as yoghurts and soups , and excluded all beverages : both energy - containing and non - energy - containing . this method has previously been used by other authors . as water accounts for much of the variability in ded , it was thought that the inclusion of beverages may disproportionately affect estimates in those with high beverage consumption . the intakes of individual nutrients were used as indicators of the nutritional quality of the diet . the intakes of macronutrients were expressed as a percentage of total energy , while micronutrients were adjusted to 10 mj / d . each of the 1945 foods ( ncfs ) and 1761 foods ( ntfs ) consumed during the surveys were aggregated into thirty - two food groups to examine the food intake patterns associated with ded . participants who consumed a nutritional supplement at least once over the recording days were defined as supplement users . weight and height measurements were taken by researchers in the participants ' own homes . weight was measured in duplicate using a seca 770 digital personal weighing scale ( chasmores ltd ) to the nearest 01 kg . height was measured to the nearest 01 cm using the leicester portable height measure ( chasmores ltd ) with the participant 's head positioned in the frankfurt plane . minimum energy intake cut - off points , calculated as multiples of bmr , were used to identify under - reporters of energy . accordingly , 32 % of children and 64 % of teenagers were classified as under - reporters . results presented below are those for the total population . as no natural cut - offs exist and ded estimates did not vary significantly with age or sex within each study , the populations were split equally into three categories : low- ( ncfs < 756 , ntfs < 765 kj / g ) , medium- ( ncfs 756875 , ntfs 766885 kj / g ) and high - ded ( ncfs > 875 , ntfs > 885 analyses of the differences between low- , medium- and high - ded groups were carried out using a one - way anova with a tukey honestly significant difference ( hsd ) or idk post hoc test to test for differences in the means . whitney u , were used when the assumptions of the anova were not met and the variables could not be normalised by transformation to the natural log or square root . the mean weight of food and of food and beverages consumed decreased across tertile of ded ( p < 0001 ) , while the mean daily intake of energy did not change significantly ( table 1 ) . table 1.daily intake of energy , macronutrients , dietary fibre and weight of food consumed , by tertile of energy density of diets in irish children ( n 594 ) and teenagers ( n 441 ) ( mean values and standard deviations)childrenteenagerslowmediumhighlowmediumhighmean dietary energy density ( kj / g)677817985663825988tertile cut - offs<756756875>875<765766885>885 n 198198198147147147mean sd mean sd mean sd p*mean sd mean sd mean sd p*weight of food excluding beverages ( g / d)845197702158603150000010554028642527312160000weight of all food and beverages ( g / d)16784141549371147336400002204791199561718525640000energy ( mj)69146915721600958125842485240116protein ( % te)14622137181252100001582514724136230000fat ( % te)32642345393474300003384935247378460000saturated fat ( % te)13625145241472600001312613825149260000monounsaturated fat ( % te)105171141711619000010921141912620000polyunsaturated fat ( % te)46114812491501155316541659190006unsaturated fat : saturated fat11402311302311502807121280291240261270280485carbohydrate ( % te)524495134552250050498524925479490007total sugar ( % te)239542364724357058420452025206490739added sugars ( % te)1244914445170580000104431264714450000starch ( % te)2754826742674801202864528143265440000dietary fibre ( g/10 mj)20241176361543800002176318744160330000%te , % total energy . arrows denote the direction of association with increasing dietary energy density . * as determined by anova . daily intake of energy , macronutrients , dietary fibre and weight of food consumed , by tertile of energy density of diets in irish children ( n 594 ) and teenagers ( n 441 ) ( mean values and standard deviations ) mean values with unlike superscript letters were significantly different between groups . arrows denote the direction of association with increasing dietary energy density . * as determined by anova . the percentage total energy ( % te ) from fat , saturated fat , monounsaturated fat and added sugars increased ( p < 0001 ) across tertile of ded , while the % te from protein and dietary fibre ( g/10 mj ) decreased ( p < 0001 ) in both populations . in teenagers alone , it was seen that as the ded increased , the % te from both carbohydrate ( p < 001 ) and starch ( p < 0001 ) decreased , while the % te from poly - unsaturated fats increased ( p < 001 ) . the % te from total sugars and the unsaturated fat : saturated fat ratio did not change significantly , in either population , as ded increased ( table 1 ) . in children , as ded increased there was a significant decrease in intakes ( energy adjusted ) of vitamin a , vitamin d , vitamin b12 , folate , biotin , thiamin , riboflavin , niacin equivalents , vitamin b6 , pantothenic acid , vitamin c , ca , mg , p , k , cu , zn , mn and na ( p < 0001 ) and fe ( p < 005 ) ( table 2 ) . table 2.daily vitamin and mineral intakes ( per 10 mj energy ) from all sources by tertile of energy density of diets in irish children ( n 594 ) and teenagers ( n 441 ) ( mean values and standard deviations)childrenteenagerslowmediumhighlowmediumhighmean dietary energy density ( kj / g)677817985663825988tertile cut - offs<756756875>875<765766885>885 n 198198198147147147mean sd mean sd mean sd p * mean sd mean sd mean sd p*vitaminsvitamin a ( g/10 mj per d)13799131015586656392000013317088884596433830000vitamin d ( g/10 mj per d)4343132242800004736262123170000thiamin ( mg/10 mj per d)25372107200600003561221320260000riboflavin ( mg/10 mj per d)2937260924080000386324142110000niacin equivalents ( mg/10 mj per d)43811340488378880000505139443102389920000vitamin b6 ( mg/10 mj per d)33382809260900004661311625090000vitamin b12 ( g/10 mj per d)694622556220000695592152230000folate ( g/10 mj per d)3631563131042828200004021893151042701060000biotin ( g/10 mj per d)4345283472730217500005246673121822922580000pantothenic acid ( mg/10 mj per d)8549742469230000927371286120000vitamin c ( mg/10 mj per d)16641291116371392153500001751593107811657555350000mineralsca ( mg/10 mj per d)12883231231314116532200001098298110229310133000011mg ( mg/10 mj per d)29843275362553800003115827840250340000p ( mg/10 mj per d)15452521472209135723300001538252144921913442200000fe ( mg/10 mj per d)13837134411304004817819215821120610000k ( mg/10 mj per d)34604543111370281939600003522537319239729124270000cu ( mg/10 mj per d)13061204100300001406120310040000zn ( mg/10 mj per d)101296248520000118441032391210000mn ( mg/10 mj per d)26092307200600003113260822080000na ( mg/10 mj per d)30535662997526285651700013150590305355829775550117 mean values with unlike superscript letters were significantly different between groups . as determined by anova , unless otherwise specified. as determined by a kruskal wallis test . daily vitamin and mineral intakes ( per 10 mj energy ) from all sources by tertile of energy density of diets in irish children ( n 594 ) and teenagers ( n 441 ) ( mean values and standard deviations ) mean values with unlike superscript letters were significantly different between groups . arrows denote the direction of association with increasing dietary energy density . * as determined by anova , unless otherwise specified . as determined by a kruskal as ded increased there was a significant decrease in intakes ( energy adjusted ) of vitamin a , folate , biotin , vitamin d , thiamin , riboflavin , niacin equivalents , vitamin b6 , vitamin b12 , pantothenic acid and vitamin c , mg , p , fe , k , cu , zn and mn decreased as ded increased ( p < 0001 ) and ca decreased ( p < 005 ) . there was no significant difference in na intakes across tertile of ded in teenagers ( table 2 ) . in both children and teenagers , however , the overall association with the micronutrient density of the diet did not change when the intake from nutritional supplements was excluded . table 3 displays the mean daily intake of food groups ( g/10 mj / d ) . fruit and vegetable intakes were inversely associated with ded in both children and teenagers ( p < 0001 ) as were fruit and vegetable juices intakes ( p < 001 ) . table 3.mean daily food group intakes ( g/10 mj ) by tertile of energy density of diets in irish children ( n 594 ) and teenagers ( n 441 ) ( mean values and standard deviations)childrenteenagerslowmediumhighlowmediumhighmean ded ( kj / g)677817985663825988tertile cut - offs<756756875>875<765766885>885 n 198198198147147147mean sd mean sd mean sd p*mean sd mean sd mean sd p*fruit and vegetablesfruit15501108992724624850000141617424925692934650000vegetables and vegetable dishes102176861142734633200001114832711524484140000fruit and vegetable juices167318111209140611481500003135617051085140680812430002cereal productswhite bread and rolls8555029294899146601626824877885078554720005wholemeal and brown bread and rolls2524351623089821300003404672093461352740000grains , rice , pasta , pizza and other cereals100480576460271865900001276109610878258377590000breakfast cereals5866134833755439102017228125385693423370000dairy productswhole milk329425193454243733825060797234123692401218224523580588reduced fat milks5341362328113628810820011515132657513339212260326cheeses1071331121321071640126141721311821311870424yoghurts7817616126243163940000396512483971322460000butter and spreadsreduced fat spreads ( < 40 % ) 2854255316470023327238222540862butter and dairy spreads ( > 40 % ) 84910087124113000089961031181331460098potatoes and potato dishespotatoes ( boiled , baked , mashed)1083794703598506499000011369158726585605150000potato products55172902019020300537220979174871960577chipped , fried and roasted potatoes47540757745366447200006225968005579277010000beveragestea and coffee435232144361298337725710188805963863604902596647960004carbonated beverages1203147716671558234623250000144517922012065322126190000diet carbonated beverages204561272671695530395218759347792859680019squashes , cordials and fruit juice drinks1039148992311471182125501024168944266854017280400meat , fish and eggsfresh meat and meat dishes10237028885696215030000158479414357359985810000burgers , sausages and meat products53539757637458139202674683656084467445020000fish and fish dishes204283109154771330000191299108264761370057eggs and egg dishes1191861141819216201641031841061711031820552confectionery and savoury snackssugars , syrups and preserves81998110795151086781126101167861310542chocolate confectionery19921322520629925400001902072201943232610000non - chocolate confectionery1542072272924429200011151851652511281780392savoury snacks1431418714321218500001171461511662192140000biscuits , cakes and pastries3042284102533802900002572942752882853140494creams , ice creams and chilled desserts44513884153233420166273642623242443260772miscellaneoussoups and sauces49958533140323429600007987784613983773830000nuts , seeds , herbs and spices1237094811350395164614106290578ded , dietary energy density . arrows denote direction of association with increasing ded . * as determined by anova , unless otherwise specified. as determined by a kruskal mean daily food group intakes ( g/10 mj ) by tertile of energy density of diets in irish children ( n 594 ) and teenagers ( n 441 ) ( mean values and standard deviations ) ded , dietary energy density . arrows denote direction of association with increasing ded . * as determined by anova , unless otherwise specified . as determined by a kruskal low ded was associated with higher intakes of wholemeal and brown bread , grains , rice , pasta , pizza and other cereals , yoghurts , potatoes , fresh meat and meat dishes , fish , soups and sauces ( p < 0001 ) , reduced fat milks , reduced fat spread ( p < 005 ) , and lower intakes of butter and dairy spreads , chipped , fried and roasted potatoes , carbonated beverages , chocolate , non - chocolate confectionery , savoury snacks and biscuits and cakes ( p < 0001 ) . in teenagers , low ded was associated with higher intakes of wholemeal and brown bread , grains , rice , pasta , pizza and other cereals , breakfast cereals , yoghurts , potatoes , fresh meat and meat dishes , soups and sauces ( p < 0001 ) , tea and coffee ( p < 001 ) and lower intakes of chips , carbonated beverages , burgers and sausages , chocolate , savoury snacks ( p < 0001 ) , white bread ( p < 001 ) and diet carbonated beverages ( p < 005 ) ( table 3 ) . with the removal of under - reporters from the analyses , mean ded was estimated for children as 826 ( 139 ) kj / g and for teenagers as 835 ( 163 ) kj / g . neither was significantly different from the total population estimates of 826 ( 144 ) kj / g for children and 826 ( 151 ) kj / g for teenagers . when analyses of associations were repeated excluding those children and teenagers who were classified as under - reporters , variations in the associations of ded with the nutritional quality of the diet were minimal . in these two national , detailed nutrition surveys of irish children and teenagers , ded was inversely associated with multiple individual markers of the nutritional quality of the diet . lower ded is generally considered to be associated with a healthier diet and it has been proposed for use as a possible proxy of dietary quality . this study adds to the evidence for the use of ded as a marker of a better nutritional quality of the diet , examining the data from children and teenagers for whom there is minimal evidence and also using dietary intake data from weighed / semi - weighed 7-d food diaries , whereas previous evidence has been from ffq and 24-h recall dietary data . in irish children and teenagers , low ded was associated with a generally better macronutrient profile : higher ( energy - adjusted ) intakes of protein and dietary fibre and lower intakes of total fat , saturated fat , monounsaturated fat and added sugars . in irish teenagers ( but not children ) , low ded was also associated with higher intakes of carbohydrates and starch and lower intakes of polyunsaturated fat . similar results were reported in swedish children and teenagers , finding that low ded was associated with higher ( energy adjusted ) intakes of carbohydrates , protein and fibre and lower intakes of total fat , saturated fat and sucrose . studies in adults have also reported low ded to be associated with higher intakes of protein , carbohydrates and dietary fibre and lower intakes of fat and saturated fat . in examining a broad range of vitamins and minerals , this study showed that lower ded was associated with a higher micronutrient density of the diet in children and teenagers . previous studies have found that children and teenagers with low ded had higher intakes of most of the micronutrients examined than those with high ded . in adults , it has been reported that those with a low ded had higher , energy - adjusted intakes of vitamins a , c and b6 , folate , fe , ca and k than their high ded counterparts and also that there were higher intakes of folate , vitamin c , carotenoids , vitamin b6 , ca and fe in those with lower ded . higher compliance with dietary recommendations for micronutrient intakes has been associated with having a lower ded . in a study of american men and women those with low ded were reported to have a higher prevalence of multivitamin / multimineral supplement use . the association of increased use of nutritional supplements with ded was also examined in the current study and was found to be associated with lower ded in irish children and teenagers . this is not to detract from the association with increased micronutrient intake , as the association of lower ded with increased micronutrient density of the diet was found to be independent of nutritional supplement usage . this better nutritional quality associated with lower ded may be explained by the differences in food intake patterns which were generally closer to healthy eating guidelines . children and teenagers with low ded , compared with those with high ded , consumed more fruit , vegetables , fruit and vegetable juices , wholemeal bread , grains , rice , pasta , pizza and other cereals , breakfast cereals ( teenagers only ) , fresh meat , potatoes ( mashed / boiled / baked ) , yoghurts and ( in children only ) fish and low - fat dairy products , and less chipped potatoes , carbonated beverages , confectionery and butter ( children only ) and ( in teenagers only ) white bread and processed meat . similar results to the current analyses were found in swedish children and teenagers ; those with lower ded were more likely to consume fruit , vegetables , pasta , rice and potatoes and cereals and less likely to consume sweets and chocolate and sweetened drinks and generally followed the nordic nutrition recommendations better than those with high ded . it had also been reported that adults consuming diets with low ded tend to be closer to dietary recommendations established by the spanish society of community nutrition than those with high ded . in a study of spanish adults , those with low ded showed a trend towards a healthier lifestyle , as characterised by more leisure - time physical activity , lower prevalence of smoking , sedentary lifestyle and alcohol consumption . some exceptions to the association of low ded with better nutritional quality of the diet were noted in this study . firstly , na intakes were slightly higher with lower ded in children but not in teenagers . these intakes , however , are estimated from food consumption data and do not take into account discretionary salt added in cooking or at the table and as such do not reflect total intakes . secondly , lower ded was associated with lower intakes of monounsaturated fat and poly - unsaturated fat ( teenagers only ) . to investigate this further the association of ded with the unsaturated fat : saturated the lack of association shown in this analysis suggests that the lower intakes of unsaturated fats seen with lower ded reflect the association of ded with lower total fat . similarly , lower unsaturated fat intakes were reported in swedish children and teenagers and spanish adults with lower ded with a decrease in the unsaturated fat - to - saturated fat ratio with increasing ded also reported for spanish adults . although some differences in the associations of ded with food intakes were noted between children and teenagers , no differences in the direction of the association of macro- and micronutrients with ded were shown , in keeping with the notion of ded as an indicator of overall dietary quality . ded is a useful marker of the nutritional quality of the diet as it is easy to calculate , is applicable to most data and can be used in the place of multiple individual markers of dietary quality or cumulative scores . one difficulty with its use is the determination of cut - off points : at what level does ded begin to reflect a less healthy diet ? this study categorised individuals according to tertile of ded but these are sample specific cut - off points and do not represent healthy and unhealthy ends of the spectrum but rather less and more healthy within the study group . this does not undermine the usefulness of ded as a marker as it can be readily used in a continuous form the cross - sectional nature of the data collection prevents the determination of the direction of associations . the percentages of under - reporters for energy in the study populations were high , particularly in teenagers ; however , upon repeating the analyses without their inclusion , the associations with a better nutritional quality of the diet remained significant . no well - accepted method for calculating the ded exists , limiting the findings of this study to the method of food only. the food - only method for calculating ded was used , as water contributes to the weight of the diet but not energy intake ; it was thought that the inclusion of beverages may have a disproportionate effect on ded and misclassify high beverage consumers ; this also allowed for comparisons with other publications . strengths of these analyses include the detailed level in which dietary intake data were collected , making use of 7-d weighed / semi - weighed diaries collected by trained nutritionists , and the use of a sample which was nationally representative of age , sex , social class , socio - economic group and geographic location . this study shows that in children and teenagers in ireland , low ded was associated with multiple individual indicators of a better nutritional quality of the diet , including higher intakes of dietary fibre and micronutrients and a generally better balance of macronutrients . low ded was also associated with food intake patterns that were generally closer to healthy eating guidelines , as well as with supplement use . taken together , these findings support the conclusion that a low ded may be a useful indicator of a better nutritional quality of the diet .

objectivesa period of thrombocytopenia is common after stem cell transplantation ( sct ) . to prevent serious bleeding complications , prophylactic platelet transfusions are administered . previous studies have shown that a rise in immature platelets precedes recovery of platelet count . our aim was to define a cutoff value for immature platelets predicting thrombopoietic recovery within 2 d.methodshematological parameters were measured on the sysmex xn hemocytometer . we calculated reference change values ( rcv ) for platelets in eight healthy individuals as marker for platelet recovery . to define a cutoff value , we performed roc analysis using data from 16 autologous sct patients.resultsrcv for platelet concentration was 14.1% . platelet recovery was observed 13 ( median ; range 931 ) days after sct . increase in immature platelet fraction ( ipf ) before platelet recovery was seen in all autologous sct patients . optimal cutoff ipf was found to be 5.3% for platelet recovery within 2 d ( specificity 0.98 , sensitivity 0.47 , positive predictive value 0.93).conclusionswe identified an optimal cutoff value for ipf 5.3% to predict platelet recovery after autologous sct within 2 d. implementing this cutoff value in transfusion strategy may reduce the number of prophylactic platelet transfusions .

for a period of 8 months ( september 2012 through april 2013 ) , eighteen adult hemato - oncological patients , scheduled for autologous sct in the maastricht university medical center , were included . the number of venous blood draws and transfusion regime were not influenced by our observational study . two patients were excluded due to missing data ( i.e. , day of platelet recovery not recorded ) . venous whole - blood samples were collected in k2-edta vacutainer tubes ( bd diagnostics , plymouth , uk ) . hemato - oncological patients admitted to the ward were routinely sampled daily around 8 am . when samples were drawn more frequently , a previous study showed that the ipf in samples with k2-edta stored at 4c stays within precision limits over 72 h 31 . whole - blood , edta anticoagulated , samples were measured on both the sysmex xe-5000 analyzer ( sysmex corporation , kobe , japan ) and the sysmex xn analyzer . earlier sysmex hematology analyzers ( such as the xe-5000 ) use the ret - channel for the measurement of immature platelets and the optic platelet count ( plt - o ) . the sysmex xn has , in addition , a novel plt - f - channel for measurement of both mature and immature platelets . this plt - f - channel was introduced to specifically gate platelets for a more accurate platelet count ( plt ) and ipf . on both analyzers , measurement of immature platelets is similar on both analyzers and is based on the principle of hemocytometry . after perforation of platelet cellular membranes by reagents , nucleic acids are stained by fluorescent dyes : polymethine and oxazine ( xe-5000 ) or oxadine ( xn ; 31,32 ) . this algorithm contains side fluorescence ( reflection of rna content ) , side scattered light ( information on intracellular structure ) , and forward scattered light ( information on cell size ) . prophylactic transfusion trigger was defined by a platelet concentration of < 10 10/l . in case of clinical complications , surgical intervention or the use of anticoagulants patients in the sct protocol received irradiated platelet products , prepared from the buffy coats of five different abo / d - matched whole - blood donors , resuspended in one unit of plasma to a volume of circa 350 ml and containing at least 250 10 platelets 33,34 . some patients received pathogen - inactivated platelets ( treated with riboflavin / uvb - pathogen ) ( mirasol ; terumobct , lakewood , co , usa ; see table2 ) . transfusion reactions were reported according to the standard protocol . to assess biological within - person variation , plt , ipf , and the absolute number of immature platelets ( ipa ) were measured in eight healthy subjects ( four men and four women , age 2333 ) once a day at the same time on 5 d in duplicate on the sysmex xn and xe analyzers . twofold nested anova was used to estimate , between - person ( cvbp ) , biological ( within - person ) ( cvwp ) , and analytical ( cva ) coefficients of variation including 95% confidence intervals 35 . rcv was calculated based on the biological and analytical coefficients of variations found in this experiment . rcv is calculated using the formula and reflects the minimal difference required to define a statistically significant increase or decrease compared with the previous result in that person . z is the number of standard deviations appropriate to the desired probability 36,37 . in this study platelet recovery was defined as the first day that platelet count increased without transfusion and exceeding the rcv . statistical analyses were performed with spss statistics version 20 ( ibm corporation , new york , ny , usa ) . sensitivity , specificity , and positive predictive value ( ppv ) were calculated as described by emir et al . 38 , where sensitivity , specificity , and ppv for our patient population are weighted averages . weights we used are based on the number of patient samples as a control ( no recovery within 2 d ) or as a case ( recovery within 2 d ) . we analyzed sensitivity and specificity of both ipa and ipf from day 8 after sct until the day of platelet recovery . using these data for a period of 8 months ( september 2012 through april 2013 ) , eighteen adult hemato - oncological patients , scheduled for autologous sct in the maastricht university medical center , were included . the number of venous blood draws and transfusion regime were not influenced by our observational study . two patients were excluded due to missing data ( i.e. , day of platelet recovery not recorded ) . venous whole - blood samples were collected in k2-edta vacutainer tubes ( bd diagnostics , plymouth , uk ) . hemato - oncological patients admitted to the ward were routinely sampled daily around 8 am . when samples were drawn more frequently , a previous study showed that the ipf in samples with k2-edta stored at 4c stays within precision limits over 72 h 31 . whole - blood , edta anticoagulated , samples were measured on both the sysmex xe-5000 analyzer ( sysmex corporation , kobe , japan ) and the sysmex xn analyzer . earlier sysmex hematology analyzers ( such as the xe-5000 ) use the ret - channel for the measurement of immature platelets and the optic platelet count ( plt - o ) . the sysmex xn has , in addition , a novel plt - f - channel for measurement of both mature and immature platelets . this plt - f - channel was introduced to specifically gate platelets for a more accurate platelet count ( plt ) and ipf . on both analyzers , measurement of immature platelets is similar on both analyzers and is based on the principle of hemocytometry . after perforation of platelet cellular membranes by reagents , nucleic acids are stained by fluorescent dyes : polymethine and oxazine ( xe-5000 ) or oxadine ( xn ; 31,32 ) . this algorithm contains side fluorescence ( reflection of rna content ) , side scattered light ( information on intracellular structure ) , and forward scattered light ( information on cell size ) . prophylactic transfusion trigger was defined by a platelet concentration of < 10 10/l . in case of clinical complications , surgical intervention or the use of anticoagulants patients in the sct protocol received irradiated platelet products , prepared from the buffy coats of five different abo / d - matched whole - blood donors , resuspended in one unit of plasma to a volume of circa 350 ml and containing at least 250 10 platelets 33,34 . some patients received pathogen - inactivated platelets ( treated with riboflavin / uvb - pathogen ) ( mirasol ; terumobct , lakewood , co , usa ; see table2 ) . to assess biological within - person variation , plt , ipf , and the absolute number of immature platelets ( ipa ) were measured in eight healthy subjects ( four men and four women , age 2333 ) once a day at the same time on 5 d in duplicate on the sysmex xn and xe analyzers . twofold nested anova was used to estimate , between - person ( cvbp ) , biological ( within - person ) ( cvwp ) , and analytical ( cva ) coefficients of variation including 95% confidence intervals 35 . rcv was calculated based on the biological and analytical coefficients of variations found in this experiment . rcv is calculated using the formula and reflects the minimal difference required to define a statistically significant increase or decrease compared with the previous result in that person . z is the number of standard deviations appropriate to the desired probability 36,37 . in this study platelet recovery was defined as the first day that platelet count increased without transfusion and exceeding the rcv . statistical analyses were performed with spss statistics version 20 ( ibm corporation , new york , ny , usa ) . sensitivity , specificity , and positive predictive value ( ppv ) were calculated as described by emir et al . 38 , where sensitivity , specificity , and ppv for our patient population are weighted averages . weights we used are based on the number of patient samples as a control ( no recovery within 2 d ) or as a case ( recovery within 2 d ) . we analyzed sensitivity and specificity of both ipa and ipf from day 8 after sct until the day of platelet recovery . using these data figure1 shows the mean concentrations and absolute ranges for plt , ipa , and the ipf of eight healthy individuals measured as described in the methods section . using these data , we calculated analytical , within - person , and between - person coefficients of variation for plt , ipa , and ipf . coefficients of variation and rcv reference change values ( rcv ) , analytical coefficient of variation ( cva , 95% confidence interval ) , within - person coefficient of variation ( cvwp , 95% confidence interval ) , and between - person coefficient of variation ( cvbp , 95% confidence interval ) for platelet count ( plt ) , immature platelet fraction ( ipf ) , and the absolute number of immature platelets ( ipa ) based on our biological variation study . mean concentrations ( dots ) and absolute range ( horizontal lines ) of platelet count ( plt ) ( panel a ) , the immature platelet fraction ( ipf ) ( panel b ) and the absolute number of immature platelets ( ipa ) ( panel c ) measured in healthy individuals ( n = 8) on 5 d in duplicate . patient characteristics aitl , angioimmunoblastic t - cell lymphoma ; beam , bcnu , cytarabine , etoposide and melphalan ; dlbcl , diffuse large b - cell lymphoma ; hdm , high - dose melphalan ; mm , multiple myeloma ; pbsct , peripheral blood stem cell transplantation . platelet recovery ( an increase in platelets greater than its rcv and not due to platelet transfusion ) was observed after a median of 13 d in patients after autologous sct . before platelet recovery , we observed an increase in ipa exceeding rcv , that is , more than 33.63% , in all patients . this increase in ipa measured on the xn analyzer preceded platelet recovery by a median of 3 d ( range 16 d ) . increase in ipf exceeding rcv , that is , more than 23.44% , was also observed in all patients prior to platelet recovery . this increase in ipf measured on the xn analyzer was seen by a median of 4 d ( range 27 d ) before platelet recovery . figure2 shows the course of plt , ipa , and ipf in a patient after autologous sct . representative example of the course of platelet count ( plt ) , the absolute number of immature platelets ( ipa ) , and immature platelet fraction ( ipf ) in a patient from stem cell transplantation ( sct ) until recovery . panel a shows the course of the plt ( black ) and the ipa ( gray ) . panel b shows the course of plt ( black ) and the ipf ( gray ) . stars indicate an increase ( in plt , ipa , or ipf ) exceeding its reference change value ( rcv ) not due to platelet transfusion . ipa and ipf , as well as xn and xe data , were analyzed separately . roc curve for ipf from day 8 till the moment of platelet recovery after autologous sct measured with the xn analyzer showed an area under curve ( auc ) of 0.86 . roc curves for the ipa , based on data from the sysmex xe-5000 , had lower aucs . aucs of roc curves based on estimated sensitivity and specificity for the ipf and the absolute number of immature platelets ( ipa ) measured on both the symex xe-5000 and the xn analyzer roc curves and aucs of immature platelet fraction ( ipf ) measured on the sysmex xe-5000 and xn analyzer after autologous stem cell transplantation ( sct ) . roc curve for ipf after autologous sct measured on the sysmex xn showed the most optimal auc . we determined a cutoff value of 5.3% [ sensitivity 0.47 ( 95% ci 0.290.65 ) , specificity 0.98 ( 95% ci 0.851.10 ) , and ppv 0.93 ( 95% ci 0.810.1.06 ) ] to predict platelet recovery within 2 d measured on the sysmex xn . figure1 shows the mean concentrations and absolute ranges for plt , ipa , and the ipf of eight healthy individuals measured as described in the methods section . using these data , we calculated analytical , within - person , and between - person coefficients of variation for plt , ipa , and ipf . coefficients of variation and rcv reference change values ( rcv ) , analytical coefficient of variation ( cva , 95% confidence interval ) , within - person coefficient of variation ( cvwp , 95% confidence interval ) , and between - person coefficient of variation ( cvbp , 95% confidence interval ) for platelet count ( plt ) , immature platelet fraction ( ipf ) , and the absolute number of immature platelets ( ipa ) based on our biological variation study . mean concentrations ( dots ) and absolute range ( horizontal lines ) of platelet count ( plt ) ( panel a ) , the immature platelet fraction ( ipf ) ( panel b ) and the absolute number of immature platelets ( ipa ) ( panel c ) measured in healthy individuals ( n = 8) on 5 d in duplicate . patient characteristics aitl , angioimmunoblastic t - cell lymphoma ; beam , bcnu , cytarabine , etoposide and melphalan ; dlbcl , diffuse large b - cell lymphoma ; hdm , high - dose melphalan ; mm , multiple myeloma ; pbsct , peripheral blood stem cell transplantation . platelet recovery ( an increase in platelets greater than its rcv and not due to platelet transfusion ) was observed after a median of 13 d in patients after autologous sct . before platelet recovery , we observed an increase in ipa exceeding rcv , that is , more than 33.63% , in all patients . this increase in ipa measured on the xn analyzer preceded platelet recovery by a median of 3 d ( range 16 d ) . increase in ipf exceeding rcv , that is , more than 23.44% , was also observed in all patients prior to platelet recovery . this increase in ipf measured on the xn analyzer was seen by a median of 4 d ( range 27 d ) before platelet recovery . figure2 shows the course of plt , ipa , and ipf in a patient after autologous sct . representative example of the course of platelet count ( plt ) , the absolute number of immature platelets ( ipa ) , and immature platelet fraction ( ipf ) in a patient from stem cell transplantation ( sct ) until recovery . panel a shows the course of the plt ( black ) and the ipa ( gray ) . panel b shows the course of plt ( black ) and the ipf ( gray ) . stars indicate an increase ( in plt , ipa , or ipf ) exceeding its reference change value ( rcv ) not due to platelet transfusion . ipa and ipf , as well as xn and xe data , were analyzed separately . roc curve for ipf from day 8 till the moment of platelet recovery after autologous sct measured with the xn analyzer showed an area under curve ( auc ) of 0.86 . roc curves for the ipa , based on data from the sysmex xe-5000 , had lower aucs . aucs of roc curves based on estimated sensitivity and specificity for the ipf and the absolute number of immature platelets ( ipa ) measured on both the symex xe-5000 and the xn analyzer roc curves and aucs of immature platelet fraction ( ipf ) measured on the sysmex xe-5000 and xn analyzer after autologous stem cell transplantation ( sct ) . roc curve for ipf after autologous sct measured on the sysmex xn showed the most optimal auc . we determined a cutoff value of 5.3% [ sensitivity 0.47 ( 95% ci 0.290.65 ) , specificity 0.98 ( 95% ci 0.851.10 ) , and ppv 0.93 ( 95% ci 0.810.1.06 ) ] to predict platelet recovery within 2 d measured on the sysmex xn . in patients awaiting thrombopoietic recovery after sct , a strategy of prophylactic platelet transfusion is considered best practice , despite questions and debates about dose and lowest acceptable platelet counts 21,22,39 . platelet transfusions , although generally accepted as safe , may cause various transfusion reactions and are considered a scarce and also costly resource 23 . in search of sensitive predictors of thrombopoietic recovery , the feasibility of immature platelets measured by the sysmex xn hematocytometer was studied as a tool to detect early platelet recruitment . an important step toward determination of a cutoff value for immature platelets is to objectively define platelet recovery . we used a validated statistical approach based on biological and analytical variation to objectify platelet recovery based on rcv 36,37 . a possible limitation may be that we assumed biological within - subject variation to be the same for healthy subjects as for patients after sct . however , this assumption appears valid because a previous study showed that for the majority of parameters , biological within - subject variation is of the same order in health and disease 40 . nevertheless , fraser 41 showed that a ratio of 0.50 of analytical variation to within - subject variation has an effect of only 12% on rcv . while within - subject variation is much larger than analytical variation in our study previous studies used different definitions for platelet recovery , for example , platelet count 30 10/l for three consecutive days , seven consecutive days with a platelet count 20 10/l without platelet transfusions , or a spontaneous increase in plt count without platelet transfusions 1315,18,28 . despite the fact that we used rcv to define platelet recovery , the interval between increase in immature platelets and platelet recovery we found appears similar , that is , approximately 2 d 15,16,18,28 . to estimate sensitivity , specificity , and ppv , we used the approach proposed by emir et al . this may be a limitation , as it led to a different number of controls per patient : more controls when recovery time is longer , which in turn led to higher specificity . day 8 was chosen because we observed earliest platelet recovery 2 d later ( day 10 ) . roc curves based on estimated sensitivity and specificity showed differences in aucs between ipf and ipa and between measurements on the sysmex xe-5000 and the sysmex xn . roc curve of ipf measured on the sysmex xn had a larger auc compared to xe-5000 . this reflects that ipf measured by the xn has a better precision than when measured by the xe-5000 . we estimated a cutoff value of ipf 5.3% in patients after autologous sct to predict thrombopoietic recovery within 2 d , with a ppv of 0.93 . based on the large between - subject variations of 32.9% and 45.9% for ipa and ipf , respectively , found in our biological variation study , it would be reasonable to define a cutoff value indicating the difference between two measurements ( or delta ) . however , roc curves based on both absolute and delta values for ipa and ipf did not have acceptable aucs , and this method was therefore not further investigated ( data not shown ) . in only one other study , a cutoff ipf predicting platelet recovery after autologous sct was estimated 18 . in this study , containing 31 patients , an ipf greater than 7.0% on day 8 after sct predicted platelet recovery within 4 d ( ppv 79% , sensitivity 76% ) . because immature platelets were assessed using flow cytometry the cutoff value for ipf of 5.3% , with high ppv and specificity , is based on observational data . theoretically , this cutoff may be used to decide whether or not to give a platelet transfusion . platelet transfusion may be omitted when platelet count is likely to recover within 2 d. this strategy may reduce the number of platelet transfusions and its associated risks and costs . the hemostatic capacity of immature platelets is a point of interest , because current prophylactic strategy is intended to prevent serious bleeding incidents . previous studies suggest that thrombocytopenic patients with high concentrations of immature platelets of up to 4050% , such as itp patients , have a smaller bleeding - tendency compared with thrombocytopenic patients with low concentrations of immature platelets , such as chemotherapy - treated patients 42 . clearly , clinical interventional studies are needed to address this issue proving more evidence for the value of high ipf in platelet transfusions strategies . another argument to include a marker of thrombopoiesis in transfusion strategy is the observation that thrombopoiesis may be suppressed by transfused platelets 7,15,27,43 . however , while previous studies reported a decrease in ipf after platelet transfusion 7,15 , we did not observe an unequivocal effect of platelet transfusions in our population . 44 showed that platelet transfusions lead to a decrease in ipf , but do not alter the ipa , suggesting dilution by an increase in circulating mature platelets . our finding was that immature platelets are sometimes increased and sometimes decreased after platelet transfusion . the dilution effect of platelet transfusion on immature platelets ( e.g. , increase or decrease ) depends on both the immature platelet count before transfusion and the immature platelet content of the platelet concentrate 17,45 . in summary , we demonstrated an increase in immature platelets measured on the sysmex xn analyzer preceding platelet recovery . we showed that ipf cutoff of 5.3% is a promising predictor of platelet recovery in patients after autologous sct . however , a small number of patients were included in the final data analysis . due to the observational design of the study interventional clinical trials in a multicenter setting are required to validate the cutoff value presented in this study and to establish its role in ipf - based transfusion policies .

background : interprofessional relationship plays a major role in effective patient care . specialized units such as critical care require multidisciplinary care where perception about every members role may affect the delivery of patient care . the objective of this study was to find out nurses perceptions of the role of physiotherapists in the critical care team.methods:qualitative study by using semi - structured interview was conducted among the qualified nurses working in the intensive care unit of a tertiary care hospital . the interview consisted of 19 questions divided into 3 sections . interviews were audio recorded and transcribed . in - depth content analysis was carried out to identify major themes in relation to the research question.results:analysis identified five major issues which included role and image of a physiotherapist , effectiveness of treatment , communications , teamwork , and interprofessional relations . physiotherapists were perceived to be an important member of the critical team with the role of mobilizing the patients . the respondents admitted that there existed limitations in interprofessional relationship.conclusion:nurses perceived the role of physiotherapist in the critical care unit as an integral part and agreed on the need for inclusion of therapist multidisciplinary critical care team .

interdisciplinary teamwork is an essential component of holistic care since team members skills , experience , and knowledge are pooled together to produce the best outcomes . each of the members in the intensive care unit ( icu ) team plays a unique role according to the patients need . physiotherapy has been accepted as an integral component of the management of patients who require intensive care and physiotherapists play a unique role as a part of the icu team . physiotherapists are elemental team representatives of the clinical healthcare team , and they need to understand other practitioners roles and communicate effectively to provide high - quality , coordinated patient care . a nurse on the other hand plays a vital role in critical care unit serves as an important communicator in the icu team . nurses make up a large component of the healthcare sector and are essential in the interprofessional healthcare team in a hospital setting . this perceived dominant role may influence the power relationship between nurses and physiotherapists . in the current demanding healthcare environment , interprofessional team practice is being promoted as a comprehensive means of providing cost - effective healthcare . literature suggests that professional specialization has led to a fragmentation between professions , which is likely to result in health care team members being unable to look at problems of patient as a whole team . a small number of studies have highlighted a part of the attitudes and perceptions that may underlie interprofessional relationships and their effect on teamwork and effectiveness of management in critical care . communication is being identified of particular interest because of the complex sociotechnical tendency of the icu environment . interpersonal factors have been reported as the main causes of stress in high - dependency areas whereas poor communication reported as cause of errors . a study exploring interprofessional perceptions of physiotherapists and midwives , using the nominal group technique and follow - up questionnaires , identified the lack of awareness about each other 's discipline . another study examined the nurse occupational therapist interface and established that there was little similarity between the work of nurses and therapists , which produced conflicts in patient handling . there stands a scarcity of literature in the indian hospital setting for incorporating interdisciplinary practice in the critical care setting and role perception between health care providers of various professions . further research about collaboration and communication between physiotherapists and nurses in the critical care unit will serve as base to improve the interprofessional relationships . the objective of this study was to examine nurses perceptions of role of physiotherapists in the critical care team at a tertiary care teaching hospital in india . this study was designed to examine nurses perceptions and experiences , rather than to measure specific identified attitudes ; semi - structured interview was used as an appropriate method . critical care nurses with the minimum experience of 6 months were recruited for the interview , after obtaining written informed consent . this study was approved by the institution ethics committee of father muller charitable institutions . a purposive this sample size was chosen to allow in - depth exploration and range of perceptions . as the study was exploratory , it did not aim to secure a strictly representative sample . a written informed consent was obtained from each participant before the commencement of the interview . investigators personally contacted the nursing supervisor from each unit to identify the total number of nurses working in that critical care team and also to recognize the ones fulfilling the inclusion criteria . once they were identified , the interviewer individually approached them and fixed up a specific time for interview according to the convenience . the interview questions were developed based on previous literature and from discussions with various experienced icu team members . the interview questions focused on the following general issues : existence of physiotherapist in the icu , the role they played , and various techniques used by the therapists while providing icu care . questions about the interaction and communication between the nurses and therapists within the icu were also asked . in addition , questions related to multidisciplinary practice and its necessity were also included to know their perceptions . although a common interview guide was used for all interviews , the precise manner in which questions were raised varied in response to the direction of each interview . in each case , however , the style of the interview was informal and unstandardized , and a nonjudgmental stance was taken to participants reported perceptions and experience . the interview took place for 1520 min in a silent room where there was no disturbance . themes emerged from the interview transcripts were categorized , and relationships between the resulting categories were identified . first , coding on a subsample of transcripts was checked by an investigator who was trained in qualitative research and having similar research interests and relevant background knowledge . through this process , thirty - three nurses ( 32 females and 1 male ) were interviewed from the critical care units . these themes , are presented under two main headings : the main issues and the key outcomes identified [ table 1 ] . principle issues from analysis the issue that emerged most strongly in relation to the physiotherapist 's role was that of facilitating ambulation in critical care patients ( n = 20 ) . all participants referred to this aspect of physiotherapy , and it was frequently placed within a functional context by reference to a range of functions . ambulation / assisting the patient to walk and limb physiotherapy ( n = 23 ) were the most commonly perceived roles of physiotherapists . the only nonphysical aspect of the physiotherapist 's role mentioned was motivating patients ( n = 1 ) . this might represent the actual role adopted by physiotherapists in the critical care team but might also reflect the way in which the other aspects of therapy are probably less visible to the health care professionals . a strong theme that emerged from the data was that how nurses perceived efficacy of physiotherapy in managing range of patients . a majority of the respondents admitted to have seen improvement in outcomes ( n = 29 ) . physiotherapy was described by several nurses as a field of practice which facilitated quicker weaning , accelerated discharge from critical unit and providing independence to patients in spite of being admitted to critical care , for example , when asked whether they had seen improvement in patients with physiotherapy ; nurse reported : yes , many number of times . mostly in case of patients ventilated . the patients actually stood up with the ventilator , and this was very surprising to see.yes , quite a lot of improvement especially after the patient has walked . so , we consider walking as a sign of shift out at times . yes , the patients actually stood up with the ventilator , and this was very surprising to see . yes , quite a lot of improvement especially after the patient has walked . we feel that they look happy and then complaint of less pain . a total number of four nurses reported that they did not notice improvement of following physiotherapy but on the other hand denied of having seen any adverse events posttherapy . when enquired about the importance of interprofessional communication , all the nurses ( n = 33 ) felt the necessity to have strong communication skills . patient 's minute details will not be missed out if good communication exists . the nature of the information exchanged between the professionals was highly valued and seen as a significant contribution to good teamwork although it appeared to be of short duration . however , some of the perceived problems in information sharing , which the nurses valued highly included lack of time : for eg . , usually we will not discuss because we will be busy with our work , but sometimes we may discuss something like if anything is to be told from physician side . interprofessional communication related to the patient care was found to be limited although there was strong felt need for interprofessional communications . all the participants felt there was frequent exchange of important information between the two professionals , especially regarding the feeding timings , sedation , and any other adverse events that occurred during the time physiotherapists were not around . in an attempt to explore any underlying conflicts , one of our questions aimed to determine the knowledge of existing disagreements between the professionals . when prompted , a few of the nurses admitted that they had minor conflicts in patient care ( n = 12 ) while a majority of them reported of not having any conflicts . the conflicts if any were resolved by simple face - to - face discussion reported by the respondents . whatever the problem was between the physiotherapist and i was settled within a day as we discussed that issue and it was never repeated again . this theorizes that selfdom or ego is not permitted in critical care as it can have a direct impact on patient 's care . certainly , the majority of the subjects admitted that there should be more interaction between nurses and physiotherapists for appropriate congruency in patient care . most of the nurses strongly felt the necessity of forming a multidisciplinary team in the critical care setting ( n = 31 ) . this was combined with considerations such as it will save each person 's time , management will be completely patient centered , and confusions will be less . these statements suggest that teamwork would thus be an accepted way of providing holistic care since team members skills , experience , and knowledge are pooled together to produce the best outcome . the issue that emerged most strongly in relation to the physiotherapist 's role was that of facilitating ambulation in critical care patients ( n = 20 ) . all participants referred to this aspect of physiotherapy , and it was frequently placed within a functional context by reference to a range of functions . ambulation / assisting the patient to walk and limb physiotherapy ( n = 23 ) were the most commonly perceived roles of physiotherapists . the only nonphysical aspect of the physiotherapist 's role mentioned was motivating patients ( n = 1 ) . this might represent the actual role adopted by physiotherapists in the critical care team but might also reflect the way in which the other aspects of therapy are probably less visible to the health care professionals . a strong theme that emerged from the data was that how nurses perceived efficacy of physiotherapy in managing range of patients . a majority of the respondents admitted to have seen improvement in outcomes ( n = 29 ) . physiotherapy was described by several nurses as a field of practice which facilitated quicker weaning , accelerated discharge from critical unit and providing independence to patients in spite of being admitted to critical care , for example , when asked whether they had seen improvement in patients with physiotherapy ; nurse reported : yes , many number of times . mostly in case of patients ventilated . the patients actually stood up with the ventilator , and this was very surprising to see.yes , quite a lot of improvement especially after the patient has walked . so , we consider walking as a sign of shift out at times . yes , the patients actually stood up with the ventilator , and this was very surprising to see . yes , quite a lot of improvement especially after the patient has walked . a total number of four nurses reported that they did not notice improvement of following physiotherapy but on the other hand denied of having seen any adverse events posttherapy . when enquired about the importance of interprofessional communication , all the nurses ( n = 33 ) felt the necessity to have strong communication skills . patient 's minute details will not be missed out if good communication exists . the nature of the information exchanged between the professionals was highly valued and seen as a significant contribution to good teamwork although it appeared to be of short duration . however , some of the perceived problems in information sharing , which the nurses valued highly included lack of time : for eg . usually we will not discuss because we will be busy with our work , but sometimes we may discuss something like if anything is to be told from physician side . interprofessional communication related to the patient care was found to be limited although there was strong felt need for interprofessional communications . all the participants felt there was frequent exchange of important information between the two professionals , especially regarding the feeding timings , sedation , and any other adverse events that occurred during the time physiotherapists were not around . in an attempt to explore any underlying conflicts , one of our questions aimed to determine the knowledge of existing disagreements between the professionals . when prompted , a few of the nurses admitted that they had minor conflicts in patient care ( n = 12 ) while a majority of them reported of not having any conflicts . the conflicts if any were resolved by simple face - to - face discussion reported by the respondents . whatever the problem was between the physiotherapist and i was settled within a day as we discussed that issue and it was never repeated again . this theorizes that selfdom or ego is not permitted in critical care as it can have a direct impact on patient 's care . certainly , the majority of the subjects admitted that there should be more interaction between nurses and physiotherapists for appropriate congruency in patient care . most of the nurses strongly felt the necessity of forming a multidisciplinary team in the critical care setting ( n = 31 ) . this was combined with considerations such as it will save each person 's time , management will be completely patient centered , and confusions will be less . these statements suggest that teamwork would thus be an accepted way of providing holistic care since team members skills , experience , and knowledge are pooled together to produce the best outcome . the issue that emerged most strongly in relation to the physiotherapist 's role was that of facilitating ambulation in critical care patients ( n = 20 ) . all participants referred to this aspect of physiotherapy , and it was frequently placed within a functional context by reference to a range of functions . ambulation / assisting the patient to walk and limb physiotherapy ( n = 23 ) were the most commonly perceived roles of physiotherapists . the only nonphysical aspect of the physiotherapist 's role mentioned was motivating patients ( n = 1 ) . this might represent the actual role adopted by physiotherapists in the critical care team but might also reflect the way in which the other aspects of therapy are probably less visible to the health care professionals . a strong theme that emerged from the data was that how nurses perceived efficacy of physiotherapy in managing range of patients . a majority of the respondents admitted to have seen improvement in outcomes ( n = 29 ) . physiotherapy was described by several nurses as a field of practice which facilitated quicker weaning , accelerated discharge from critical unit and providing independence to patients in spite of being admitted to critical care , for example , when asked whether they had seen improvement in patients with physiotherapy ; nurse reported : yes , many number of times . mostly in case of patients ventilated . the patients actually stood up with the ventilator , and this was very surprising to see.yes , quite a lot of improvement especially after the patient has walked . so , we consider walking as a sign of shift out at times . yes , the patients actually stood up with the ventilator , and this was very surprising to see . yes , quite a lot of improvement especially after the patient has walked . a total number of four nurses reported that they did not notice improvement of following physiotherapy but on the other hand denied of having seen any adverse events posttherapy . when enquired about the importance of interprofessional communication , all the nurses ( n = 33 ) felt the necessity to have strong communication skills . patient 's minute details will not be missed out if good communication exists . the nature of the information exchanged between the professionals was highly valued and seen as a significant contribution to good teamwork although it appeared to be of short duration . however , some of the perceived problems in information sharing , which the nurses valued highly included lack of time : for eg . , usually we will not discuss because we will be busy with our work , but sometimes we may discuss something like if anything is to be told from physician side . interprofessional communication related to the patient care was found to be limited although there was strong felt need for interprofessional communications . all the participants felt there was frequent exchange of important information between the two professionals , especially regarding the feeding timings , sedation , and any other adverse events that occurred during the time physiotherapists were not around . in an attempt to explore any underlying conflicts , one of our questions aimed to determine the knowledge of existing disagreements between the professionals . when prompted , a few of the nurses admitted that they had minor conflicts in patient care ( n = 12 ) while a majority of them reported of not having any conflicts . the conflicts if any were resolved by simple face - to - face discussion reported by the respondents . whatever the problem was between the physiotherapist and i was settled within a day as we discussed that issue and it was never repeated again . this theorizes that selfdom or ego is not permitted in critical care as it can have a direct impact on patient 's care . certainly , the majority of the subjects admitted that there should be more interaction between nurses and physiotherapists for appropriate congruency in patient care . most of the nurses strongly felt the necessity of forming a multidisciplinary team in the critical care setting ( n = 31 ) . this was combined with considerations such as it will save each person 's time , management will be completely patient centered , and confusions will be less . these statements suggest that teamwork would thus be an accepted way of providing holistic care since team members skills , experience , and knowledge are pooled together to produce the best outcome . the major role of a physiotherapist as perceived by the nurses was facilitating early mobilization . this could be attributed to the fact in the center where this study was carried out practices early mobilization protocol which was designed and tested indigenously . most of the respondents felt that a physiotherapist could help in case of ventilated patients , thereby reducing the chances of ventilated associated infections and improving patients condition and promote quicker recovery . all the nurses commented on the need to have good communication in the critical care unit . a study of this kind advocated that communication would be a significant step in assisting healthcare delivery to clients and their families to become flawless , efficient , and effective . hence , there is need for the same to reflect a core competency between health professionals . a majority of nurses felt lack of time as the major reason for lack of communication in the critical care practice . disagreements are another criterion which requires to be pondered on when there is any discussion on critical care . some of the respondents felt that disagreements are bound to take place in a setting where two professions do not work in congruency . hence the major factor that may influence this is interprofessional learning , and it has shown to have a positive impact on their awareness and understanding of other professional roles , interprofessional issues , and how they valued others roles as well as the support they offer , which in turn helped to reduce barriers and interprofessional conflicts . perhaps understandably , as each profession views the issue from its own perspective , the blame for change or adaptation is liable to be shifted to the other profession . this suggests that if change is to be effective , it should be bilateral , with the blame for change shared across the two professions . one way in which this might occur would be through nurses and physiotherapists working concurrently with the patient , accomplishing the goals of both nursing and therapy in the same activities . for example , a change of the patient 's position to side lying may simultaneously provide skin pressure care and improve lung ventilation and sputum clearance . it would appear that physiotherapists should consider spending some time sharing with nurses sufficient skills to provide continuity of care for patients . physiotherapists can not on one hand expect nurses to provide continuity in progression and on the other hand be protective of therapy skills in this area . in any case , physiotherapists should not be unduly worried on this score . brown and greenwood suggest that such flexible cooperation might enhance the core skills of each profession by promoting the working roles of both . nurses also , despite the problems identified , spoke of good or excellent relationships with physiotherapists but admitted that it lacked teamwork because they did not discuss patient goals with each other often , so they did not work together on strategies to achieve those goals . one of the major limitations of this study is the smaller sample size which might not be able derive the perception of entire nurse population . this preliminary exploratory study revealed that nurses working in the critical care team have positive perception about the physiotherapists . participants of this study were aware of the role of physiotherapists in critical care , which will be useful in clinical decision making of the therapist .

these are peer - reviewed poster - platform submissions finalized by the scientific program committee . a total of 153 abstracts ( 14 platforms [ pp1 through pp14 ] & 139 posters [ 1 through 139 ] ) were selected from 161 submissions to be considered for presentation during november 4 8 , 2011 , at the hilton baltimore hotel , to pathologists , cytopathologists , cytotechnologists , residents , fellows , students , and other members of cytopathology - related medical and scientific fields .

introduction . the aim of this study was to determine the accuracy of linear measurements in dry human skulls in ideal position and different deviated positions of the skull . methods . 6 dry human skulls were included in the study . opaque markers were attached to alveolar bone . buccolingual and mesiodistal distances and heights were measured in 5 different regions of either jaws using a digital caliper . radiographic distances were measured in ideal , rotation , tilt , flexion , and extension positions of the skulls . the physical and radiographic measurements were compared to estimate linear measurement accuracy . results . the mean difference between physical measurements and radiographic measurements was 0.05 0.45 . there was a significant difference between physical measurements and radiographic measurements in ideal , rotation , tilt , and extension positions ( p value < 0.05 ) . conclusions . the accuracy of measurements in galileous cbct machine varies when the position of the skull deviates from ideal ; however , the differences are not clinically significant .

cone beam computed tomography ( cbct ) has found its niche in different fields of dental practice during recent years [ 15 ] . nowadays , the use of three - dimensional radiographies is increasing for diagnosis and treatment in dentistry . single - slice ct and multislice ct techniques were first introduced in this regard [ 6 , 7 ] . the case against these two systems was that they exposed patients to high - radiation doses [ 810 ] . thus cbct was introduced to promise low - radiation doses together with adequate image quality , as well as fast image processing and lower costs [ 11 , 12 ] . consequently , the use of this technique dramatically increased in implant dentistry , maxillofacial surgery [ 1 , 13 ] , orthodontics , endodontics , and so forth . as different treatment approaches highly depend on the exact estimation of distance between anatomical landmarks and bone thickness , many clinicians tend to use the linear measurement capability of cbct . unfortunately , unwanted measurement errors may lead to catastrophic consequences like treatment failure [ 16 , 17 ] . since cbct machines are not equipped with cephalostat , the skull might be in eccentric position during scanning procedure . previous studies have investigated accuracy of linear measurements in cbct images using the newtom 3 g , accuitomo , and other cbct machines [ 17 , 1922 ] , concluding that the linear measurement capability of these units is reliable for the structures closely associated with dentomaxillofacial imaging . have evaluated the effect of patient 's head position on linear measurement accuracy of newtom 3 g cbct machine . mischkowski et al . evaluated the geometric accuracy of scans obtained with galileous cbct device . they declared the liner measurements between anatomical structures in the present of soft tissue are significantly accurate . although the newly introduced galileous cbct machine is reported to be one of cbct dental devices with the lowest effective dose , its linear measurement accuracy has not been evaluated in different positions . since it is probable that the patient 's head deviate from true vertical or horizontal orientation during scanning procedure ; which might adversely affect measurement accuracy , the aim of this study was to determine the accuracy of linear measurements in dry human skulls in ideal position and different deviated positions by simulating clinical relevant distances , using galileous cbct machine . this is an experimental study on human skulls which was conducted in isfahan university of medical sciences and approved by ethical committee of isfahan dental school research center . 5 regions were selected on each jaw ; anterior , premolar , and molar regions of left and right sides of the jaw . in order to measure buccolingual and mesiodistal distances and height in each region , four points were determined using 1.5 mm rod - shaped gutta - percha ( size # 40 ) opaque markers ( table 1 ) : in the way that the first marker was glued to the embrasure of buccal alveolar crest of the studied region , the second marker to embrasure of lingual alveolar crest , perpendicular to the first marker , the third marker to the most apical region of buccal alveolar crest in the same direction with the first marker , and the fourth marker to the adjacent tooth 's embrasure of buccal alveolar crest , next to the first marker . the buccolingual , mesiodistal distances and heights of each region were measured two times by the first observer with one - week interval and once by a second observer , using a digital caliper ( guanglu , taizhou , china ) . the images were taken using galileos comfort 3d imaging system ( sirona dental systems inc . 5 different radiographs were provided for each skull in different positions : ideal position and positions with 10-to-15-degree rotation , 10-to-15-degree tilt , 10-to-15-degree forward tilt ( flexion ) , and 10-to-15-degree backward tilt ( extension ) . to reconstruct the temporomandibular joint space , a 1.5 mm - thick baseplate wax was placed between condylar process and temporal fossa . after guiding the jaw into the centric occlusion , the jaws were attached by an adhesive tape . a polyvinyl pipe was placed into the foramen magnum and attached to a camera tripod ( zeiss universal tripod ft6302 , oberkochen , germany ) with capability of lateral and forward - backward tilt and rotational adjustment , via a dial plate . to provide standard radiographs , the skull was held in the image field using the machine 's occlusal bite block between teeth ( in the way that the occlusal plane was perfectly horizontal ; according to manufacturer 's instructions ) . to ensure appropriate position of skulls , the system 's light localizer , which displays the midsagittal line , then , imaging was performed at 7 ma ( 42 mas ) and 85 kvp , with 14-second scan time and 270 rotation . each scan produced 200 projections in a 15 15 15 cm field of view . a charge - coupled device detector , with 1024 1024 matrix and 0.15 mm voxel size , was used to detect the images . afterwards , the radiographic distances were measured twice by the first observer with two - week interval and once by the second observer . it has to be added that the measured distance was from the end of one marker to the end of another . the physical and radiographic distances , measured in each region , are illustrated in table 1 . intraclass correlation coefficient ( icc ) was used to analyze intraobserver and interobserver reliability of measurements ( = 0.05 ) . wilcoxon test was used to compare physical and radiographic values of different measurements ( = 0.05 ) ; the less the differences between physical and radiographic measurements are , the more accuracy of radiographic measurements will be . one - sample t - test was used to compare the differences with the acceptable 0.5 mm mean absolute error ( = 0.05 ) . univariate analysis of variance was used to assess the difference between radiographic and physical measurements , considering the confounding variables . due to severe bone resorption , the distances in the mandibular left premolar and molar regions in one skull and also mandibular right premolar and molar regions in another skull were not separately measured , reducing the whole measurements to 174 . according to icc values , interobserver correlations for radiographic measurement and for physical measurements were both 0.996 ( p value < 0.001 ) . the mean difference between physical measurements and radiographic measurements in the present study was 0.05 0.45 . there was a significant difference between physical measurements and radiographic measurements in ideal , rotation , tilt , and extension positions . accuracy of measurements for height was significantly lower than physical measurements in all positions ( p value < 0.05 ) . tukey hsd showed that the accuracy of measurements on skull number 5 was significantly lower than skulls number 2 , 3 , 4 and 6 ( p value = 0.022 , 0.001 , 0.006 , and 0.001 , resp . ) . also , accuracy of measurements in rotation and flexion positions was significantly lower than ideal position ( p value = 0.042 , 0.043 resp . ) . there was no significant difference among accuracy of measurements in molar , premolar , and central regions ( p value < 0.05 ) . accuracy of measurements in the left sides of skulls was significantly lower than the right side ( p value = 0.01 ) . there was no significant difference between upper and lower jaws in terms of accuracy of measurements ( p value = 0.115 ) . table 5 compares the absolute mean difference between each radiographic position and 0.5 mm acceptable error . this table shows that the accuracy of measurements in all cases was above 0.5 mm . it is important to determine whether the accuracy of measurements decreases or remains unchanged , when the patient 's head position changes . the aim of this study was to determine the accuracy of linear measurements in dry human skulls in different positions of the skull by simulating clinical relevant distances . in the present study , soft tissue was not simulated to prevent its probable confounding effect on accuracy of measurements . main errors in patient 's head position ( tilt , extension , flexion , and rotation ) were evaluated . lateral displacement was not assessed , since this position error less likely occurs . due to the probable effect of teeth situation and the type of jaw , both mandible and maxilla radiographic units should be able to measure height , mesiodistal length , and buccolingual length of implant treatments , all of these distances were included in the present study . the rod - shaped opaque markers would let the observers more easily and accurately measure the distance between the end of one marker and the end of another . to make sure that the measurements were accurate , two observers measured the distances . the intraobserver and inter - observer correlations above 0.95 show that the accuracy was acceptable . the results of the study showed that there were some differences in measurement between normal and deviated positions in some cases ; however , since the average difference was less than 0.5 mm , they are not considered clinically significant . comparison of the accuracy of radiographic measurements between ideal and deviated positions showed that significant difference exists in rotation and flexion positions , which may suggest that these positions are the most effective deviations for measurement accuracy . hassan et al . , in assessment of accuracy of measurements on dry human skulls , found no significant difference between different positions of skulls . this contrast with the present study is probably due to lower number of measurements and longer distances in that study . this contrast might be due to different distances that were measured in these studies , as well as different study designs . the high rates of standard deviation of differences between physical and radiographic measurements in the present study suggest that more than a few factors are affecting the accuracy of measurements . there are a number of reasons that may justify differences in the accuracy of measurements . predominant artifacts in cbct imaging including noise , scatter , extinction artifact , beam hardening , exponential edge gradient effect , aliasing artifacts , and ring artifacts may cause difficulties in detecting the exact situation of objects in a cbct output image which leads to inaccurate measurements . moreover , the anatomical distortions , a function of shape and orientation of the structures , can cause distortions as well . results of the present study showed that accuracy of measurements is affected by skull type . this suggests that cbct units can be enhanced by features like a posterior position adjustment , letting the practitioner consider anatomical asymmetries and differences ; however , distortion caused by anatomical asymmetry is not always distinguishable from radiographic distortion . on the other hand , although some deviated skulls showed severe distortion in the panorama view of galaxis software in the present study , the distances between markers could still be accurately measured in cross - sectional , tangential , and axial views . this is partly due to the fact that cbct software lets the practitioner choose not only the orientation of reconstructed image layer but also the image plane that cross - sectional , tangential , and axial views are expected to display ; however , failure in appropriate setting will lead to difficulties in measuring distances . for instance , to see acceptable cross - sectional image planes , one should previously set the image plane in panorama view . if the practitioner fails to do so , cross - sectional display will not be appropriate for measurements . as an observation of the present study , sometimes the markers that were precisely placed in buccal and lingual embrasures of a tooth could not be displayed at the same time in cross - sectional view of the software , even with changing the image plane in panorama or the orientation of reconstructed image layer , which implies a shortcoming in the unit or the software and should be corrected . the mean difference between physical measurements and radiographic measurements in the present study was 0.05 0.45 mm . ludlow et al . who used the same method for selection of the points , reported 0.29 0.20 mm mean difference . marmulla et al . however , used computer algorithm to localize measurement points with pixel fractionalization . these differences are due in part to the different study designs , measurement techniques , and cbct units used . moreover , leung et al . who measured alveolar bone height reported that the accuracy was 0.6 mm . these contrary results can be explained by the fact that determining anatomical landmarks is more difficult than opaque markers . the mean absolute error of galileos cbct machine was estimated to be 0.26 0.18 mm by mischkowski et al . . the difference between this study and the present study is probably due to different investigated areas and distances measured . lund et al . reported that the measurements in cbct were very accurate and the absolute mean difference was even less than voxel size . in fact , lund et al . used an object consisting of plexiglass plates , while the present study was performed on human dry skulls . in the present study for evaluating the effect of different head position all skulls it may be the reason of significant difference of between the left and right side . the manufacturer of galileos cbct machine claims that the accuracy of length measurements is 0.15 mm ; however the results of the present study showed that this accuracy depends on numerous criteria and is not always in the above range . we suggest that the same study with larger number of skulls be conducted so that more criteria such as aspect of measurement can be included in univariate anova . moreover , it is recommended to simulate soft tissue attenuation in one separate group to assess its probable effect . in conclusion , the present study demonstrates that the accuracy of measurements in galileos cbct machine varies when the position of skull deviates from ideal ; however the reduction in accuracy could be clinically considered insignificant .

pure red cell aplasia is characterized as a normocytic anemia associated with reticulocytopenia and the absence of erythroblasts in the bone marrow . pure red cell aplasia can be induced by various causes such as thymoma , connective tissue disease , viral infection , lymphoma , and adverse drug reactions . there have been only a few reports of pure red cell aplasia associated with acute viral hepatitis a. in korea , no case of pure red cell aplasia caused by acute hepatitis a has yet been reported . we recently experienced a case of acute viral hepatitis a complicated by pure red cell aplasia . the patient was successfully treated with corticosteroids . here we report this case and review the literature .

pure red cell aplasia ( prca ) is a disorder that was first described in 1922 and that is characterized as a normocytic anemia associated with the absence of erythroblasts in the bone marrow . prca can be induced by various causes.1 however , only a few cases of prca associated with hepatitis a have been reported.2 - 6 in korea , there have been several reports of prca associated with thymoma , lymphoma , sle , hashimoto 's disease , parvovirus , and adverse drug reactions . however , no case of prca caused by acute hepatitis a has yet been reported . in this report , we describe a case of prca with acute hepatitis a and review the literature . a 36-year - old male was admitted with a 3-day history of fever , jaundice , and vomiting . his vital signs were as follows : blood pressure , 120/90 mmhg ; heart rate , 88 beats / min ; respiratory rate , 18/min ; and body temperature , 37.1. he had obvious jaundice and icteric sclera . his hemoglobin concentration was 17.1 g / dl , his red cell count was 5.7410/l , his white blood cell count was 5,000/l , and his platelet count was 114,000/l . blood chemistry analyses revealed a total bilirubin level of 3.8 mg / dl ; direct bilirubin , 2.39 mg / dl ; ast , 7736 iu / l ; alt , 3558 iu / l ; ldh , 14,407 iu / l ; albumin , 3.8 g / dl ; prothrombin time , 18.6 s ( 10.1 - 12.4 s ) ; bun , 50.9 mg / dl ; and cr , 5.7 mg / dl . tests for hepatitis b surface antigen , and igm anti - hepatitis b core , and anti - hepatitis c virus antibody were all negative . tests for human immunodeficiency virus antibody and epstein - barr virus vca igm antibody were negative and parvovirus was undetectable by polymerase chain reaction . c3 was 86.6 mg / dl , c4 was 53 mg / dl , igg was 746 mg / dl , iga was 442 mg / dl , and igm was 469 mg / dl . the results of a chest x - ray , abdominal ultrasonography , and duodenoscopy were unremarkable . he received hemodialysis treatment owing to acute renal failure three times a week from day 2 . on day 4 , he was diagnosed with hepatitis a and treated with fluid therapy . on admission , there was no sign of anemia , but his hemoglobin level had gradually decreased . , his hemoglobin level had dropped to 4.8 g / dl and his reticulocyte count had significantly decreased to 0.13% . the bone marrow biopsy showed 50% cellularity and the myeloid : erythroid ratio was 16.1:1 . a paucity of erythroid precursors was noted , although granulopoiesis was normal in number and maturation . differential erythroid cell counts of aspirates were as follows : pronormoblast , 1.4% ; basophilic normoblast , 2.4% ; polychromatic normoblast , 0.0% ; and orthochromatic normoblast , 0.0% . as a result of these findings , he was diagnosed with pure red cell aplasia associated with acute hepatitis a. he began treatment with intravenous methylprednisolone ( 1 mg / kg / day ) daily at day 29 . after the steroid therapy , his hemoglobin level and renal function began to improve steadily . hemodialysis was stopped on day 34 , and the hemoglobin level had increased to 8.3 g / dl on discharge . one month after discharge , the prednisolone dose was tapered to 10 mg in the outpatient clinic . on day 48 after discharge , prednisolone treatment was stopped , because the hemoglobin level had improved to 13.9 g / dl and the cr level had decreased to 1.3 mg / dl ( fig . prca results in an anemia associated with severe reticulocytopenia and the absence of marrow erythroid precursor cells.1 innate prca is known as blackfan - diamond anemia , and is usually diagnosed among infants less than 1 year of age . in adults , prca usually occurs as an acquired disease , mainly due to thymoma , connective tissue disease , viral infection , lymphoma , and adverse drug reactions.1 in the present case , while the patient was being treated with fluid therapy and hemodialysis for hepatitis a with acute renal failure , his hemoglobin level gradually decreased . no signs of gastrointestinal bleeding were observed and no evidence of thymoma was apparent in the chest x - ray . results were negative for human immunodeficiency virus antibody , epstein - barr virus vca igm antibody , viral hepatitis b antibody , viral hepatitis c antibody , anti - nuclear antibody , parvovirus pcr , and rheumatoid arthritis factor . there were no suspicious findings for lymphoma in the peripheral blood smear or bone marrow . bone marrow examination showed marked erythroid hypoplasia with normal granulocytes and megakaryocytes . as a result , he was diagnosed with prca caused by acute hepatitis a. globally , cases of acute hepatitis a associated with prca are very rare . in all of the cases reported to date , transient , severe anemia appeared after the acute phase of the hepatitis had passed and disappeared within a short period after blood transfusion or corticosteroid therapy . the clinical course of prca with hepatitis a and the prognosis of the patients were good.2 - 6 the mechanism of prca seen in many cases of viral hepatitis is poorly understood . bone marrow and liver contain components of the reticuloendothelial system and may , therefore , be adversely affected by similar agents . previously known main mechanisms are viral - induced autoimmune response and direct viral infection of bone marrow progenitor cells.1 the autoimmune response can directly cause a cytolytic reaction to bone marrow progenitor cells by t - cells , natural killer cells , or complement binding with antibody and can damage bone marrow stromal cells.1 idiopathic prca can be initially treated with corticosteroids and blood transfusion . generally , the recovery period is about 1 month.7 in a study of 27 prca patients using corticosteroids for 2 to 5 weeks , remission occurred within 4 weeks in 40% of cases , and a cure rate of 37% was reported.8 case studies of prca with acute viral hepatitis are rare ; cases of treatment with corticosteroids and blood transfusion have been reported.2 - 6 in corticosteroid - resistant cases , prca can be treated with cyclosporin a or cyclophosphamide . a study of 43 patients treated with cyclosporin a showed an overall response of 65%.9 one study reported that the duration of remission induced by cyclophosphamide seemed to be prolonged compared with that induced by corticosteroid.8 in addition , several studies reported that antithymocyte globulin , alemtuzumab , and rituximab are useful in the treatment of refractory prca.7 in summary , we have reported the first case of prca associated with acute hepatitis a in korea . the patient showed an early response to corticosteroids and successfully achieved remission after 2 months of treatment .

genes that increase susceptibility to age - related macular degeneration ( amd ) have been identified ; however , since many individuals carrying these risk alleles do not develop disease , other contributors are involved . one additional factor , long implicated in the pathogenesis of amd , is the lipofuscin of retinal pigment epithelium ( rpe ) . the fluorophores that constitute rpe lipofuscin also serve as a source of autofluorescence ( af ) that can be imaged by confocal laser ophthalmoscopy . the af originating from lipofuscin is excited by the delivery of short wavelength ( sw ) light . a second autofluorescence is emitted from the melanin of rpe ( and choroid ) upon near - infrared ( nir - af ) excitation . sw - af imaging is currently used in the clinical management of retinal disorders and the advantages of nir - af are increasingly recognized . here we visit the damaging properties of rpe lipofuscin that could be significant when expressed on a background of genetic susceptibility . to advance interpretations of disease - related patterns of fundus af in amd , we also consider the photochemical and spectrophotometric features of the lipofuscin compounds responsible for generating the fluorescence emission .

age - related macular degeneration ( amd ) is a complex disorder that is influenced by genetic and environmental factors ; heritability is estimated to account for 45% to 71% of cases [ 1 , 2 ] . several ( ~20 ) amd genetic susceptibility genes have been identified with two loci , complement factor h ( cfh ( 1q32 ) and age - related maculopathy susceptibility 2/htra serine peptidase 1 ( arms2/htra1 ) on 10q26 accounting for 50% of amd cases [ 3 , 4 ] . the cfh locus harbors many independent risk and protective haplotypes [ 5 , 6 ] while for arms2/htra1 a single major risk haplotype is associated with amd . multiple studies have also demonstrated that haplotype - tagging single - nucleotide polymorphisms ( snps ) in cfh and arms2 , are major determinants of amd endophenotypes and disease progression . specifically , cfh - rs1061170 is associated with drusen and with both early and advanced amd while arms2-rs10490924 is strongly associated with reticular pseudodrusen and rapid progression to late amd [ 5 , 79 ] . nevertheless , by various estimates , currently known genetic loci account for only 50%75% of overall amd risk . interest in a role for retinal pigment epithelium ( rpe ) lipofuscin in amd stems from the knowledge that it accumulates with age [ 10 , 11 ] , is high in central retina , exhibits behaviors toxic to rpe [ 1215 ] and exhibits a link to drusen formation . advances in non - invasive fundus imaging have facilitated the diagnosis and differentiation of retinal disease . in vivo imaging provides a window within which to view the natural course of retinal disease . of the available imaging modalities , fundus autofluorescence ( af ) has proven to be especially valuable , in large part because disease - related processes can alter the distribution of the af signal . the natural autofluorescence of the fundus that is excited by sw light ( 488 nm excitation ) ( figure 1 ) exhibits spectral features and an age - relationship that indicates a principle origin from the fluorescent pigments that accumulate in rpe cells as lipofuscin . unlike lipofuscin species that accumulate in other non - dividing cells , the pigments of rpe lipofuscin are produced in the membranes of photoreceptor outer segments from non - enzymatic reactions of vitamin a aldehyde [ 1821 ] . this fluorescent material is transferred to rpe cells within phagocytosed outer segment disks [ 22 , 23 ] and becomes deposited in the lysosomal compartment of the cells . in the healthy retina , the age - related increase levels off after age 70 perhaps because of a loss of photoreceptor or rpe cells and/or changes in fluorescence emission due to extensive photooxidation / photodegradation of the bisretinoid compounds ( discussed below ) . rpe lipofuscin consists of a complex mixture of fluorophores that have been identified in by chromatography and mass spectrometry and characterized structurally ; all of the known bisretinoid lipofuscin pigments have been detected in human eyes ( figure 2 ) . ( a2-gpe ) , a2e and isomers of a2e [ 2836 ] , dimers of all - trans - retinal having a cyclohexadiene head group ( all - trans - retinal dimer ) [ 33 , 37 ] and the associated protonated schiff base conjugate and the uncharged a2-dhp - pe ( a2-dihydropyridine - phosphatidylethanolamine ) . higher molecular weight adducts also form when aldehyde - bearing cleavage products of bisretinoid react with intact bisretinoid molecules . other molecular constituents of rpe lipofuscin are adducts of cep ( 2-(-carboxyethyl)-pyrrole ) , hne ( 4-hydroxynonenal ) and mda ( malondialdehyde ) that are derived from oxidative fragmentation of lipid . products of lipid oxidation are generally non - fluorescent or blue - emitting fluorophores [ 42 , 43 ] and in this case could be generated by the photoreactivity of other lipofuscin fluorophores . accumulation of bisretinoids in rpe cells is unlikely to depend on an inhibition of lysosomal enzyme activity , since this fluorescent material is amassed in all healthy eyes beginning at early ages . . they present with a central six - carbon ring from which extend two polyene arms terminating in ionone rings . each of these arms is derived from a molecule of retinaldehyde and constitutes a separate light - absorbing chromophore , one arm absorbing in the ultraviolet range and the other in the visible ( figure 2 ) . the numbers of alternating carbon - carbon double and single bonds that form the conjugation systems of the arms determine the wavelength of absorbance ; the longer conjugation system in each molecule confers absorbance in the visible range . absorbances in the visible spectrum are significant since these wavelengths reach the retina . since these adducts of retinaldehyde are held together by covalent bonds , bisretinoids do not provide stores of retinoid for the visual cycle , as has been suggested . in clinical settings , sw - fundus af is excited by wavelengths ranging from 488 nm , the excitation employed with a confocal scanning laser ophthalmoscope ( cslo ) , to the 535580 nm range utilized by a modified fundus camera and the 568 nm light used with fluorescence adaptive optics ophthalmoscopy [ 17 , 47 , 48 ] . fundus autofluorescence measured in vivo by spectrophotometry has a broad excitation spectrum that peaks between 490510 nm . the fluorescence emission is also broad and centered at approximately 600 nm [ 11 , 17 ] . rpe lipofuscin ex vivo exhibits an excitation spectrum that peaks between 450490 nm ; the fluorescence emission is maximal at ~600 nm ( figure 3 ) . moreover , just as with fundus autofluorescence , the emission spectrum recorded from whole lipofuscin exhibits red - shifts when excited by progressively longer wavelengths ( figure 3 ) . thus the spectral characteristics of fundus autofluorescence are consistent with that of rpe lipofuscin [ 43 , 5052 ] and chiefly with an origin from the bisretinoid fluorescent pigments that are known constituents of rpe lipofuscin . the bisretinoids that have been characterized have absorbance maxima varying from 440 nm to 510 nm and they emit with an orange fluorescence that peaks at ~600 nm . the bisretinoid a2e can emit fluorescence at longer wavelength excitations such as 545 nm ( figure 3b ) . while there exists no evidence that bisretinoids of rpe lipofuscin can undergo lysosomal degradation , loss of this material due to photodegradation has been demonstrated . thus studies of rpe lipofuscin [ 5355 ] and individual bisretinoid lipofuscin fluorophores such as a2-gpe , all - trans - retinal dimer and a2e have revealed that these compounds are photoinducible generators of reactive oxygen species such as singlet oxygen and superoxide anion . singlet oxygen in turn reacts with the conjugated double bond systems comprising the arms of the bisretinoid molecules [ 5661 ] leading to the fragmentation of the parent molecules and release of aldehyde - bearing cleavage products such as methylgloxal and glyoxal that can react with and inactivate proteins . these small dicarbonyls also provoke the formation of advanced glycation end products ( age ) that deposit extracellularly [ 16 , 62 ] . ages incite inflammatory processes and since they are detected in drusen [ 63 , 64 ] , they reflect a link between rpe bisretinoid lipofuscin and the formation of sub - rpe deposits . photooxidation of a2e and all - trans - retinal dimer has also been shown to incite complement activation [ 65 , 66 ] . photooxidation is clearly an ongoing process in the eye since photooxidized forms of a2e and all - trans - retinal dimer have been detected in isolated human and mouse rpe [ 25 , 57 ] . these processes likely contribute to bruch s membrane thickening and photoreceptor cell degeneration [ 68 , 69 ] in abca4 mutant mice and are a cause of the increased vulnerability of albino abca4 mice to retinal light damage . the propensity for bisretinoids to undergo photooxidative and photodegradative processes may underlie the decline in rpe lipofuscin fluorescence emission ( photobleaching ) that has been observed in non - human primates during in vivo fluorescence imaging by adaptive optics scanning laser ophthalmoscopy [ 48 , 71 ] , with cell culture models and in non - cellular assays ( figure 4 ) . lipofuscin photobleaching may also explain why after surgical repair of some cases of retinal detachment , hyperautofluorescent lines coursing parallel to retinal blood vessels can be visible in fundus af images [ 73 , 74 ] ( figure 5 ) . the hyperautofluorescent imprint has been interpreted as indicating a change in the position of the vessel relative to the underlying retinal tissue and is visible because of contrasting levels of af brightness . at any given time , the intensity of fundus af is likely the difference between fluorophore synthesis on the one hand , and lipofuscin photoxidation / photodegradation in rpe , on the other . under the shadow of a blood vessel , the formation of bisretinoid from retinaldehyde ( with 11-cis being converted to all - trans - retinal ) would likely continue unabated [ 20 , 30 , 7577 ] but lipofuscin photooxidation and photobleaching would be substantially reduced . as a result , a vessel imprint of more intense af would be revealed upon retinal translocation . when rpe lipofuscin is assayed by recording fluorescence in histological sections of human retina , the signal is found to increase from central fovea to perifovea and after peaking at an eccentricity of ~8 , it decreases towards the periphery [ 10 , 78 ] . a similar pattern has been observed with quantitative fundus autofluorescence ( qaf ) ( figure 6 ) ; at an eccentricity of 10 , qaf is approximately 95% of that measured centrally . reduced foveal fundus autofluorescence is due in large part to absorption of the exciting light by macular pigment and to the higher optical density of melanin in central rpe . by fundus spectrophotometry , qaf and fluorescence photomicroscopy , the highest levels of rpe lipofucin in healthy eyes unexpectedly , this pattern was not replicated in another study relying on fluorescence measurements in flat - mounted human cadaver eyes . based on the spatial distribution of mass peaks detected with matrix - assisted laser desorption - ionization imaging mass spectrometry ( maldi - ms ) investigators have reported that a2e is detectable in central retina of mice , but not in the central human rpe . instead , the highest a2e levels were restricted to a small patch of rpe found exclusively in the far periphery of temporal retina . this patch was not matched by similar a2e signal in in the periphery of superior , nasal or inferior retina . while a2e is well known to be fluorescent , surprisingly , this patch was almost devoid of fluorescence . since the spectral features of rpe lipofuscin and fundus autofluorescence are best accounted for by the excitation and emission spectra of the bisretinoid constituents , the fluorescence originating in centrally situated rpe cells likely originates from some combination of these di - retinal adducts . interestingly , the maldi - ms findings could indicate that the various species of bisretinoid lipofuscin compounds exhibit spatial heterogeneity . reduced levels of a2e in the macula could also be a consequence of greater lipofuscin photocleavage in central rpe ; an explanation such as this could account for the greater susceptibility of the macula to disease . failure to detect a2e centrally , may even be attributable to the limitations of the methodology . maldi - ms is a surface - based technique that depends on the extraction of analyte into a matrix applied to the surface of the tissue . factors that could cause spatial differences in extraction efficiency are regional differences in rpe height and spatial differences in the compartmentalization of lipofuscin . for instance rpe cells are taller and narrower in the human macula with melanin being uppermost and lipofuscin at greater depths in the cells . after age 50 complex organelles containing both melanin and lipofuscin ( melanolipofuscin ) predominate in the cells with macular rpe containing more melanolipofuscin than rpe at the equator and periphery . thus it is likely that the extraction from central rpe is less efficient because of the depths of lipofuscin in the cells and/or difficulty in accessing lipofuscin from complex melanolipofuscin - organelles . the healthy fundus also exhibits a near - infrared autofluorescence ( nir - af ) ( > 800 nm ) when excited at ~787 nm [ 84 , 85 ] ( figure 1 ) . the intensity of the fundus nir - af is at least 60 times less than sw - af . several lines of evidence indicate that rpe and choroidal melanin serve as a source of the nir - af signal . additionally , the high nir - af signal at the fovea corresponds to the elevated optical density of melanin in this area . conversely , nir - af emanating from a full - thickness macular hole is similar in brightness to surrounding retina . the more frequent use of sw - af may be due , in part , to the introduction of the heidelberg spectralis having optical coherence tomography ( oct ) capability ( hra + oct ) and to the subsequent decline in the popularity of the hra2 and spectralis hra in retinal clinics . the oct module in the spectralis reduces nir - af signal intensity , thus compromising nir - af image quality as compared to cslos without oct ( e.g. , hra2 and spectralis hra ) . nevertheless , nir - af has advantages over sw - af . for instance , during image acquisition , patients are not disturbed by the nir - af light as they are with the sw - af exciting light . in the presence of retinal disease such as amd , patterns and intensities of fundus autofluorescence are notably altered [ 8893 ] . at locations of rpe and photoreceptor cell demise ( atrophy ) both the sw - af and nir - af signals become strikingly deficient or absent [ 94 , 95 ] . these areas of atrophy can take the form of discrete spots , isolated patches or large expanses ( geographic atrophy , ga , usa ) . while ga presents as areas of darkness in both sw and nir - af ( figure 7 ) , the lesion size can sometimes appear larger with either the sw - af or nir - af modality [ 84 , 97 ] ( figure 7 ) . nevertheless , the rate of increase in ga area is the same whether measured in sw- or nir - af images . in exudative amd , the developing neovascular lesion can be discerned in sw - af images early on as a focal hyperautofluorescence . later the sw - af signal is reduced within the lesion [ 99 , 100 ] . in some cases the edge of the neovascular lesion exhibits increased sw - af signal . in the zone of retina immediately adjacent to geographic atrophy , there are often additional af changes . these aberrant signals can present as intermittent foci or continuous bands of altered brightness in both sw - af and nir - af images [ 101103 ] . in these junctional zones areas of increased sw - af can coincide with increased nir - af ; increased sw - af can overlap with reduced nir - af ; or nir - af signal can be enhanced while sw - af may appear normal [ 84 , 104 ] ( figure 7 ) . interestingly , a loss of photoreceptor function was found to be associated with both increased and decreased nir - af [ 97 , 105 ] . since both sw - af and nir - af are considered to originate in rpe cells , it is puzzling that sw - af can be increased at positions where nir - af is reduced or absent . this apparent incongruity has been attributed to abnormal rpe cells that have lost melanin while accumulating excessive levels of lipofuscin due to accelerated rates of outer segment phagocytosis [ 84 , 105 ] . the lipofuscin forms in photoreceptor cells prior to disc shedding and phagocytosis . as is evident from studies of the rcs rat , the fluorophores of lipofuscin form and accumulate in photoreceptor outer segment debris even in the absence of phagocytosis [ 106108 ] . other observations indicate that photoreceptor cells are likely to be degenerating in these junctional zones of increased sw - af . in particular , retinal sensitivity , measured by microperimetry , is commonly reduced at positions presenting with increased sw - af as compared to normal sw - af [ 105 , 109 ] . in addition , oct findings in the zone of enhanced autofluorescence surrounding ga include thinning of the reflective band attributable to rpe / bruch s membrane and disruption of the reflectivity band corresponding to the ellipsoid zone of photoreceptor inner segments [ 96 , 110 ] thus could it be that at positions of diminished nir - af , rpe cells are atrophied or lost and impaired photoreceptor cells become a source of accelerated lipofuscin formation and thus enhanced sw - af ? mechanistically , mishandling of retinaldehyde , the precursor of lipofuscin , is known to lead to elevated bisretinoid formation in photoreceptor cells and compromised photoreceptor cells may not be able to provide the energy needed for reduction of retinaldehyde to the non - reactive alcohol form . similar mechanisms may explain the rapid onset of elevated fundus sw - af that co - localizes with scotomas associated with acute macular neuroretinopathy ( amn ) [ 93 , 112 ] . the observation that nir - af signals increase in parallel with increases in sw - af at the border of ga may also not be fully understand . hyperautofluorescence foci at the junctional zone of ga , at least in some cases , can be attributed to abnormally superimposed rpe cells [ 113 , 114 ] . but whether this can account for more extensive bands of high nir - af is not certain . it has been suggested that rpe lipofuscin may contribute to nir - af of the fundus . however , albino rats do not exhibit nir - af despite the presence of lipofuscin and as shown in figure 8 , we have not detected an nir - af signal from synthesized samples of a2e that otherwise emit fluorescence when excited at 488 nm . nir - af emission from other bisretinoids is unlikely , given the small structural differences amongst these compounds ( figure 2 ) . if melanogenesis is the basis for increased nir - af brightness at the border of ga , as has been suggested [ 84 , 105 ] , one might expect that increased melanin concentrations would be visible as hyperpigmented spots and bands in color fundus photographs . in considering an optical effect as an explanation for increased nir - af signal it could be that rpe lipofuscin does not fluoresce in the nir range but can modify the nir - af emission from melanin . this could occur if lipofuscin secondary lysosomes intercalate amongst apically situated melanosomes [ 78 , 116 ] thereby reducing the nir - af quenching associated with secondary self - absorbance of the fluorescence emission . a mechanism such as this might also explain why hyperautofluorescent rings visible in the nir - af fundus images in retinitis pigmentosa exhibit spatial correspondence with high intensity rings observed in sw - af images . interest in a role for rpe lipofuscin in amd stems from its age - related increase [ 10 , 11 ] , an accumulation that is more pronounced in central retina , a propensity for adverse effects on rpe and photoreceptor cells [ 1215 , 68 , 70 ] and links to drusen formation [ 16 , 67 ] . contributions to amd susceptibility from rpe lipofuscin would exist within the context of background genetic risk . since the products of bisretinoid photodegradation can be damaging [ 16 , 60 ] , it is worth considering whether lipofuscin lost by photooxidation / photodegradation is more significant than the lipofuscin remaining in the cells . at any given time , the lipofuscin in rpe that is recorded by sw - af may consist of only some portion of the fluorescent material that has been accumulated over a life - time . sw - af emitted from rpe bisretinoids is commonly regarded as a way to monitor the health of the rpe , with areas of high af indicating increased lipofuscin levels and areas of low af indicating rpe loss . however , as discussed here there is increasing evidence that interpretations of the sw - af signal are complex . for instance , impaired photoreceptors may generate increased levels of bisretinoid fluorophores thus amplifying sw - af signal . ultimately , an understanding of patterns of fundus af will impact the use of these images to assess therapeutic outcomes . while fundus autofluorescence provides en face spatial information , the spectral features of the fluorescence are not elucidated and the cellular origin of the fluorescence is not identified . recently , this approach has been shown to enable the differentiation of similar phenotypes having disparate genetic origins [ 44 , 118 ] . the qaf approach may eventually help to ascertain the role of lipofuscin in various retinal disorders including amd . because of more limited gradations of signal and thus greater contrast , diseased versus non - diseased areas of retina are easier to distinguish in nir - af images as compared to sw - af images . indeed , in recessive stargardt disease ( stgd1 ) , sw - af changes are often not obvious at fundus locations where abnormalities are detectable in nir - af images . in addition , in many cases the low nir - af signal corresponds spatially to loss of the inner segment ellipsoid zone ( ez ) in spectral domain ( sd ) oct images . while the origin of the nir - af signal may not be completely understood , the nir - af signal can provide a good estimate of the size of geographic atrophy and surrounding abnormalities . all of these issues favor the inclusion of nir - af in the management of retinal diseases such as amd .

recent studies suggest that aldh1 is a putative marker for hnscc - derived cancer stem cells . however , the regulation mechanisms that maintain the stemness and metastatic capability of hnscc - aldh1 + cells remain unclear . initially , hnscc - aldh1 + cells from hnscc patient showed cancer stemness properties , and high expression of bmi1 and snail . functionally , tumorigenic properties of hnscc - aldh1 + cells could be downregulated by knockdown of bmi-1 . overexpression of bmi-1 altered in expression property aldh1 cells to that of aldh1 + cells . furthermore , knockdown of bmi-1 enhanced the radiosensitivity of radiation - treated hnscc - aldh1 + cells . moreover , overexpression of bmi-1 in hnscc - aldh1 cells increased tumor volume and number of pulmonary metastatic lesions by xenotransplant assay . importantly , knock - down of bmi1 in hnscc - aldh1 + cells significantly decreased distant metastases in the lungs . clinically , coexpression of bmi-1/snail / aldh1 predicted the worst prognosis in hnscc patients . collectively , our data suggested that bmi-1 plays a key role in regulating snail expression and cancer stemness properties of hnscc - aldh1 + cells .

head and neck squamous cell carcinoma ( hnscc ) , including oral squamous cell carcinoma ( oscc ) , is the sixth most prevalent type of malignancy worldwide and accounts for approximately 8% to 10% of all cancers in southeast asia [ 1 , 2 ] . hnscc - related mortality is mainly caused by cervical lymph node metastasis , and occasionally by distant organ metastasis . the epithelial - mesenchymal transition ( emt ) is a process in which epithelial cells lose their polarity and adopt a mesenchymal phenotype . this process is thought to be a critical step in the induction of tumor metastasis and malignancy . mani et al . demonstrated that induction of emt results in cells that have stem cell properties and generates cells with properties similar to breast cancer stem cells . snail , a member of the zinc - finger transcription factor family , is one of the master regulators that promotes emt and mediates invasiveness as well as metastasis in many different types of malignant tumors [ 7 , 8 ] . the aldehyde dehydrogenase ( aldh ) family of enzymes is comprised of cytosolic isoenzymes that oxidize intracellular aldehydes and contribute to the oxidation of retinol to retinoic acid in early stem cell differentiation . recently , aldh has been reported to be a unique marker of head and neck cancer stem cells ( csc ) [ 10 , 11 ] . aldh1 was also found to co - localize with other cscs - related markers , including mmp-9 , cd44 , and ck14 , at the invasive front of the tumor . these hnscc - aldh1 cells displayed the radioresistance and represented a reservoir of cells that have the proliferative potential to generate tumors . these findings suggested that snail expression may regulate the tumorigenesis , radiochemoresistance , and cancer stem cell properties of malignant hnscc tumors . however , the molecular mechanisms involved in mediating metastasis and tumor malignancy of hnscc - csc through the regulation of snail remain unknown . bmi-1 is a member of the polycomb ( pcg ) family of transcriptional repressors that mediate gene silencing by regulating chromatin structure . bmi-1 is essential for maintaining the ability of neural , hematopoietic , and intestinal stem cells to self - renew [ 1517 ] . bmi-1 was identified as a proto - oncogene that cooperates with myc to promote the generation of lymphoma . additionally , bmi-1 has been verified as a predictor of prognosis in bladder cancer , prostate cancer , brain cancer [ 22 , 23 ] , breast cancer , pancreatic cancer , and lung cancer . bmi-1 has been demonstrated to play a role in the tumorigenesis of hnscc [ 27 , 28 ] . bmi-1 has also been reported to be involved in tumor metastasis [ 29 , 30 ] . recently , an elegant study by song et al . showed that bmi-1 can directly promote emt and malignancy in nasopharyngeal carcinoma by regulating snail . the goal of this study was to clarify the relationship between bmi-1 , snail , and aldh1 in hnscc or hnscc - associated csc and the involved molecular mechanisms . the study was approved by the institutional ethics committee / institutional review board of taipei veterans general hospital . the dissociated cells from the samples of hnscc patients were suspended at 1 10 cells / ml in 37c dmem supplemented with 2% fcs . the identification of aldehyde dehydrogenase 1 ( aldh1 ) positive hnscc cells was carried out using the aldefluor assay ( stemcell technologies , durham , nc , usa ) and fluorescence - activated cell sorting . cells were suspended in aldefluor assay buffer containing aldh substrate ( baaa , 1 mol / l per 1 10 cells ) and incubated for 40 min at 37c . as a negative control , for each sample of cells , an aliquot was treated with 50 mmol / l diethylaminobenzaldehyde ( deab ) , a specific aldh inhibitor . the sorting gates were established using the cells stained with pi only as a negative control ; the aldefluor - stained cells treated with deab and staining with a secondary antibody alone to test for viability . hnscc - aldh1 cells were cultured in a medium consisting of serum - free dmem / f12 ( gibco - brl , gaithersburg , md ) , n2 supplement ( r and d systems inc . , minneapolis ) , 10 ng / ml bfgf ( r and d systems ) , and 10 ng / ml egf ( r and d systems ) [ 13 , 32 ] . briefly , total rna ( 1 g ) of each sample was reverse - transcribed using 0.5 g oligo dt and 200 u superscript ii rt ( invitrogen ) . the primer sequences for real - time rt - pcr were listed in table 2 . the amplification was carried out in a total volume of 20 l containing 0.5 moll of each primer , 4 mmoll mgcl2 , 2 l lightcyclertm - faststart dna master sybr green i ( roche molecular systems , alameda , ca ) , and 2 l of 1 : 10 diluted cdna . pcr reactions were prepared in duplicate and performed using the following program : 95c for 10 min , followed by 40 cycles of denaturation at 95c for 10 sec , annealing at 55c for 5 sec , and extension at 72c for 20 sec . standard curves ( cycle threshold values versus template concentration ) were prepared for each target gene and for the endogenous reference gene ( gapdh ) for each sample . quantification of unknown samples was performed using lightcycler relative quantification software version 3.3 ( roche ) . the oligonucleotide 5-aaaacctaatactttccagattgatttggat ccaaatcaatctggaaagtattagg-3 targeting human bmi-1 ( nm_005180 , nt 10611081 ) was synthesized and cloned into plvrnai to generate the lentiviral expression vector , plvrnai / sh - bmi1 . the lentiviral expression vector carrying bmi-1 full - length cdna , plv / bmi-1 was obtained from biosettia inc . pcmvr8.9 and pmd.g , expressing gag - pol and the vesicular stomatitis virus envelope , respectively , were provided by the consortium ( academia sinica , taipei , taiwan ) . the lentiviruses were generated by cotransfecting 5 10 293 ft cells per 10 cm plate with lentiviral vector and packaging plasmids using lipofectamine 2000 ( lf2000 , invitrogen ) . subconfluent cells were infected with lentivirus at a multiplicity of infection of 5 in the presence of 8 g / ml polybrene ( sigma - aldrich ) [ 13 , 33 ] . total rna was extracted from cells using trizol reagent ( life technologies , bethesda , md , usa ) and the qiagen rnaeasy ( qiagen , valencia , ca , usa ) column for purification . affymetrix hg u133 plus 2.0 microarrays containing 54,675 probe sets for > 47,000 transcripts and variants , including 38,500 human genes . a typical probeset contains eleven 25-mer oligo nucleotide pairs ( a perfect match and a mismatch control ) . for microarray analysis , sample labeling , hybridization , and staining the average linkage distance was used to assess the similarity between two groups of gene expression profiles as described below . the difference in distance between two groups of sample expression profiles to a third was assessed by comparing the corresponding average linkage distances ( the mean of all pairwise distances ( linkages ) between members of the two groups concerned ) . the error of such a comparison was estimated by combining the standard errors ( the standard deviation of pairwise linkages divided by the square root of the number of linkages ) of the average linkage distances involved . classical multidimensional scaling ( mds ) was performed using the standard function of the r program to provide a visual impression of how the various sample groups are related . all procedures involving animals were in accordance with the institutional animal welfare guidelines of taipei veterans general hospital . eight - week - old scid mice and/or nude mice ( balb / c strain ) were injected with 105 cells orthotopically . in vivo gfp imaging was performed using an illuminating device ( lt-9500 illumatool / tls equipped with an excitation source ( 470 nm ) and filter plate ( 515 nm ) ) . tumor size was measured with calipers and the tumor volume was calculated using the formula ( length width2)/2 . the integrated optical density of green fluorescence intensity was captured and analyzed using image pro - plus software [ 33 , 34 ] . the statistical package of social sciences software ( spss , inc . , chicago , il ) was used for statistical analysis . an independent student 's t - test was used to compare the continuous variables between groups . a log - rank test was used to compare the cumulative survival durations in different patient groups . initially , parental , isolated aldh1 , and aldh1 cells were isolated from tissue samples of six hnscc patients using the aldefluor assay and the fluorescence - activated cell sorting ( facs ) analysis ( figure 1(a ) and table 1 ) [ 13 , 35 ] . it has been reported that cancer stem - like cells can be cultured in suspension to generate floating spheroid - like bodies ( sb ) under serum - free medium with bfgf and egf . interestingly , aldh1 increased higher tumor spheres - forming capability than that of aldh1 ( figure 1(b ) ) . furthermore , aldh1-derived spheres with regular 10% serum cultivation increased epithelial - attached cells and differentiation marker ( ck18)(see figure 1(a ) in supplementary material available online at doi : 10.1155/2011/609259).to evaluate the enhancement of tumorigenicity of hnscc - aldh1 cells , soft agar colony formation assays and matrigel / transwell - invasion and were examined . compared with parental and aldh1 , aldh1 derived from hnscc patients no.1 and no . 2 showed colony - forming ability and higher invasion activity ( figures 1(c ) and 1(d ) ) . to evaluate the in vivo tumor initiating capability of aldh1 and aldh1 , we injected 1000 , 3000 , and 10 cells into the neck of scid mice . the results showed that 104 aldh1 did not induce tumor formation but 3,000 aldh1 from the hnscc tissues of six patients in xenotransplanted mice all resulted in the generation of visible tumors 6 weeks after injection ( table 1).the results of xenotransplanted analysis further showed that aldh1 demonstrated higher abilities to induce tumor growth ( figure 1(e ) ) . lastly , serial xenotransplanted analysis suggested that aldh1 had in vivo self - renewal ability ( supplementary figure 1(b ) ) . based on these findings , the aldh1-lineage cells isolated from hnscc patients presented the significant tumor - initiating abilities , especially in aldh1 cells from patients no . 1 and no real - time rt - pcr data demonstrated that the stemness and emt - related genes ( especially in bmi-1 and snail ) were significantly activated in hnscc aldh1 ( table 2 and data not shown ) . to further investigate the role of bmi-1 in maintaining the biological properties of hnscc - aldh1 , we used a loss - of - function approach , in which bmi-1 was knocked down by small hairpin rna ( shrna ) in hnscc - aldh1 cells . stable knockdown of bmi-1 in hnscc - aldh1 cells was achieved by transduction with lentivirus that expressed shrna targeting bmi-1 ( sh - bmi-1 ) . lentivirus that expressed shrna targeted against luciferase ( sh - luc . ) was used as a control . western blot analysis confirmed that shbmi-1 repressed bmi-1 protein expression in hnscc - aldh1 cells ( figure 2(a ) ) . importantly , silencing bmi-1 expression led to downregulation of snail and aldh1 expression ( figure 2(a ) ) . additionally , our results showed that silencing of bmi-1 in hnscc - aldh1 cells inhibited the ability of the cells to form colonies on soft agar ( figure 2(b ) ) and migrate / invade ( figure 2(c ) ) . to evaluate whether overexpression of bmi-1 could enhance the tumorigenic properties of hnscc - aldh1 cells , we generated stable bmi-1-overexpressing ( bmi-1over ) hnsccs using lentiviral transduction ( figure 2(d ) ) . total proteins from hnscc - aldh1 overexpressing bmi-1 exhibited elevated expression of snail and aldh1 ( figure 2(d ) ) . in addition , overexpression of bmi-1 significantly increased soft agar colony formation ( figure 2(e ) ) , and migration / invasion of hnscc - aldh cells ( figure 2(f ) ) . taken together , our results suggest that bmi-1 modulates the in vitro tumorigenic properties in hnscc - aldh1 or aldh1 cells by regulating snail . to explore molecules governing stemness and tumorigenicity in hnscc - cd44aldh1 cells treated with bmi1-overexpressing lentivirus , we examined their transcriptome profile using gene expression microarray analysis ( figure 3(a ) ) . principle component analysis ( pca ) further showed that the transcriptome profile of hnscc - aldh1 cells overexpressing bmi-1 demonstrated higher expression levels of embryonic stem cells ( escs ) transcriptomes ( table 3 and figure 3(b ) ) . multidimensional scaling analysis further demonstrated that hnscc - aldh1 cells and hnscc - aldh1 cells overexpressing bmi-1 are more similar to escs than hnscc - aldh1 cells ( * p < .05 ; figure 3(c ) ) . to validate the microarray analysis results , real - time pcr was performed to confirm that the mrna expression levels of the embryonic genes ( oct-4 , nanog , sox2 , klf4 , and lin28 ) , emt - related genes ( snail and slug ) , and drug - resistant - related genes ( mdr-1 and abcg2 ) in bmi-1-overexpressing aldh1 cells were significantly higher than those in aldh1 cells ( * p < .05 ; table 2 and figure 3(d ) ) . we next sought to determine if bmi-1 expression could modulate the in vivo tumor initiating activity in immunocompromised nude mice . to monitor the in vivo growth of aldh1 , aldh1 , and bmi-1-overexpressing aldh1 cells , these cells were transfected using a lentivector combined with the green fluorescent protein gene ( gfp ) and followed by in vivo gfp imaging system . firstly , the results showed that 1 10aldh1 cells did not induce tumor formation in nude mice , but 1000 aldh1 cells generated visible tumors 6 weeks after injection ( table 1 ) . in contrast to aldh1 cells , one of three ( 33.3% ) nude mice was detected with the tumor formation after 6-week transplantation of 3000 bmi-1-overexpressing aldh1 cells . furthermore , tumor volumes in hnscc - aldh1 transplanted mice were significantly decreased when mice were treated with sh - bmi-1 ( table 1 ; figure 4(a ) ) . overexpression of bmi-1 enhanced in vivo tumor growth in hnscc - aldh1 ( table 1 ; figure 4(a ) ) . furthermore , we investigated the role of bmi-1 in the radio sensitivity of hnscc - aldh1 and hnscc - aldh1 treated with sh - bmi-1 and bmi-1 overexpressing . an ionizing radiation ( ir ) dose of 0 to 10 gy was applied to these cells , and hnscc - aldh1 cells showed greater radioresistance than the aldh1 cells ( p < .05 ; figure 4(b ) ) . knockdown of bmi-1 in aldh1 cells results in significant inhibition of radioresistance while overexpression of bmi-1 in aldh- cells promotes radioresistant properties ( p < .05 ; figure 4(b ) ) . moreover , to confirm that bmi-1 is crucial for metastasis in vivo , mice were injected with different numbers of aldh1 , aldh1/sh - bmi-1 , aldh1/bmi-1over or control gfp - expressing aldh1 cells . 5x105 bmi-1-overexpressing aldh1 cells significantly increased local invasion , distant metastasis to the lungs and tumor size compared with control aldh1 cells ( figures 5(a ) and 5(b ) ) . in addition , silencing bmi-1 in aldh1 cells effectively reduced the number of lung metastases and tumor size in vivo ( figures 5(a ) and 5(b ) ) . taken together , our results reveal a crucial role for bmi-1 signaling in the maintenance of in vivo tumorigenicity and metastasis of hnscc - aldh1 and -aldh1 cells . elevated snail protein expression in hnscc is correlated with the development of metastasis and poor survival . elevated expression of aldh1 also correlates with poor prognosis for hnscc patients . to investigate whether there is a positive correlation between bmi-1 , snail , and aldh1 in head and neck cancers , we studied the expression of bmi-1 , snail , and aldh1 by immunohistochemical ( ihc ) staining of a panel of specimens array from 93 hnscc patients . the ihc results showed that elevated expression of bmi-1 , snail , and aldh1 was positively associated with high - grade , poorly differentiated hnscc ( figure 6(a ) ) . our results also showed a significant positive correlation between aldh-1 , bmi-1 ( figure 6(b ) ) ; aldh-1 and snail ( figure 6(c ) ) ; bmi-1 and snail ( figure 6(d ) ) in hnscc tissues . this is consistent with previous studies that reported that hnscc - aldh1 cells have elevated bmi-1 and snail expression [ 13 , 38 ] . to determine the prognostic significance of bmi-1 , snail , and aldh1 coexpression in patients with hnscc patients who were triple positive for bmi-1 , snail , and aldh1 were predicted to have the worst survival rate compared with other head and neck cancer patients ( figure 6(e ) ; bmi-1/snail / aldh1 versus other groups ) . overall , these data indicate that expression of bmi-1 , snail , and aldh1 in hnscc patients could be a critical factor in predicting disease progression and clinical outcomes . a recent study demonstrated that bmi-1 mrna and protein overexpressed in a subpopulation of tumor initiating cells in cd44 + hnscc , which possessed self - renewal and tumor formation ability . zhang et al . also reported that there are side populations of oral squamous cell carcinomas that express high levels of abcg2 , abcb1 , cd44 , oct-4 , bmi-1 , nspc1 , and ck19 . our previous work showed that hnscc - aldh1 + cells have high levels of bmi-1 . the ability to self - renew and radiochemoresistance confirmed that aldh1 + -lineage cells underwent epithelial - mesenchymal transition ( emt ) and endogenously co - expressed snail . in the current study , our data demonstrated that hnscc - aldh1 cells had high levels of bmi-1 , at both the mrna and protein levels ( figure 2 ) . using a lentiviral vector expressing shrna targeting bmi-1 , we observed that the level of aldh1 expression and tumorigenic properties of hnscc - aldh1 could be down - regulated by knockdown of bmi-1 ( figure 2 ) . importantly , overexpression of bmi-1 could turn hnscc - aldh1 into cancer stem cell - like hnscc - aldh1 cells ( figure 3 ) . consistent with these findings , the immunohistochemical survey of 93 hnscc patient tissues showed a positive correlation between expression of bmi-1 , snail , or aldh1 and tumor stage ( figure 6 ) . kaplan - meier analysis demonstrated that patients expressing bmi-1 , snail , and aldh1 were predicted to have the worst survival prognosis of hnscc patients ( figure 6(e ) ) . however , a recent study showed a significant correlation between negative bmi-1 protein expression and the recurrence of tongue cancer . tongue cancer patients , especially female tongue cancer patients , usually do not have these habits . the close relationship between tongue cancer and human papillomavirus has been explored by many researchers [ 4245 ] . the prognosis of hnscc patients with distant metastases in the lung , liver , and bone is very poor [ 3 , 46 ] . in this study , we found that bmi-1 can regulate snail and aldh1 ; change the emt - related genotypes of the aldh1 cells ; and modulate distant lung metastases ( figure 5 ) . distant metastases have been reported to be associated with bmi-1 expression in breast cancer [ 4749 ] , melanoma , gastric cancer , and colon cancer . microarray analysis revealed that eleven gene signatures , which were correlated to the bmi-1-driven pathway , were closely related to distant lung metastases . bmi-1 is the target gene of sall4 in human hematopoietic as well as leukemic cells and is down - regulated if sall4 is knocked down by the sirna in the hl-60 leukemia cell line [ 52 , 53 ] . recently , researchers employed microrna profiling to gain insight into the role of bmi-1 in regulating emt . overexpression of mir-200c decreased bmi-1 expression in breast cancer stem cells ( bcscs ) and inhibited the formation of mammary ducts as well as tumors by normal mammary stem cells and bcscs . found that mir-15a and mir-16 directly targeted the bmi-1 3 untranslated region and correlated with bmi-1 protein levels in ovarian cancer patients and cell lines . together , our research shows that the bmi-1 signaling pathways play a major role in the maintenance of stemness and the metastatic ability of hnscc - csc by regulating of snail expression . additionally , we demonstrate coexpression of bmi-1 , snail , and aldh1 in hnscc patients

neuroleptic malignant syndrome ( nms ) is a life - threatening emergency that is often seen as a complication of antipsychotic agents . it is characterized by a tetrad of motor , behavioral , autonomic , and laboratory abnormalities . we report a case of a 34-year - old man with a history of newly diagnosed type 2 diabetes mellitus , mental retardation , and behavioral abnormalities who developed nms after starting on antipsychotic agents . he presented with high temperature , muscle rigidity , tachycardia , and elevated blood pressure . after a week of hospital treatment in the general ward of a secondary care unit , he was discharged in a hemodynamically and mentally stable state .

neuroleptic malignant syndrome ( nms ) is a rare but life - threatening complication to antipsychotic agents . it was initially reported in 1960s and since then many cases have been recognized worldwide . nms is associated with a significant mortality rate and requires early recognition and prompt treatment . early diagnosis of nms can be a clinical challenge to family physicians who treat patients with both medical and psychiatric conditions . with increasing the use of antipsychotic agents in primary and secondary care setting , nms can be missed if not suspected in patients on antipsychotics presenting in general practice with hyperthermia and muscle rigidity . we report a case of nms diagnosed in a patient with poorly controlled diabetes in the urban health center at vellore , tamil nadu , india . a 34-year - old male with history of type 2 diabetes mellitus on oral hypoglycemic agents was hospitalized twice with symptoms of ketosis . poor drug compliance , poor motivation regarding diabetes care , and psychomotor agitation were noted during hospitalization . one week after starting on antipsychotics , he presented with extrapyramidal symptoms of dystonia , parkinsonian gait , fine tremors , high spiking fever , altered sensorium , and muscle rigidity . blood counts , urine microscopy , and renal function were normal except for low sodium [ table 1 ] . creatine phosphokinase ( cpk ) was ordered in view of muscle rigidity that was very high ( 1543 ) . a diagnosis of nms was made according to diagnostic and statistical manual of mental disorders ( dsm - iv ) criteria . laboratory abnormalities in our patient risperidone was stopped immediately , and he was treated with lorazepam , trihexyphenidyl , paracetamol , and intravenous fluids in consultation with psychiatrist . within 48 h of hospitalization , family was informed of the diagnosis and the need for close monitoring of his glycemic control . nms is a rare condition as reported in the two studies done in neurology and psychiatric units of teaching hospitals in india with an incidence of 1.401.41/1000 cases treated with antipsychotics . among the risk factors , some studies report older age as high - risk due to the associated medical morbidities , nutritional deficiencies , dehydration and electrolyte abnormalities , male gender , and genetic predisposition . other studies report more cases in the age group of 2050 years associated with high antipsychotic dosage as seen in our patient . the antidopaminergic activity of antipsychotic drugs is associated with the symptoms of muscle rigidity , hyperthermia , autonomic dysfunction , and mental status change . these symptoms are recognized as diagnostic according to the american psychiatric associations dsm - iv . the signs and symptoms develop during a 2472 h period following the administration of antipsychotic drug ; however , it can develop later as seen in our patient . newer antipsychotic agents such as risperidone block serotonin receptors more than dopamine receptors ; however , nms has been reported with their use as in our case study . anti - emetics such as metoclopramide and droperidol have been linked to nms by their dopaminergic blocking activity . laboratory abnormalities may include leukocytosis , electrolyte disturbances , and elevated cpk secondary to muscle damage . diagnostic tests for fever may include urine analysis , chest radiography , and lumbar puncture . imaging studies of brain are not diagnostic of nms , however , may rule out other causes of altered mental status . possible complications include dehydration from poor oral intake , renal failure secondary to rhabdomyolysis , and coagulation abnormalities . mortality in nms has decreased from 76% to between 10% and 20% , however , complete recovery is noted in most patients . mortality is caused by complications such as respiratory failure , cardiovascular collapse , renal failure , arrhythmias , and thromboembolism . patients who are hemodynamically unstable are to be transferred to higher centers for intensive monitoring . mild cases can be managed at the secondary care setting in consultation with a psychiatrist . supportive therapy involves discontinuation of antipsychotic agents , correction of electrolyte imbalances , nutritional deficiencies and monitoring of airway , breathing , and circulation . our patient was managed at the urban health center by the team of family physicians . specific dopaminergic agents such as bromocriptine , dantrolene , and electroconvulsive therapy as an option are considered for more severe cases by psychiatrists . symptoms may last for a month in patients who were on depot preparations . restarting antipsychotics in patients with history of nms if needed is done on consultation with psychiatrist . depot preparations are generally not recommended , however , a 2 weeks interval is to be considered between recovery and restarting antipsychotic agents . early detection and management of side effects caused by neuroleptic agents are of particular consideration to family physicians who attend to the early symptoms . decreasing risk factors that aggravate rigidity include avoiding dehydration , minimal use of restraints and intramuscular injections , adequate nutrition , screening for history of nms in the patient , or other family members .

chronic myeloid leukemia ( cml ) is a myeloproliferative neoplasm characterized by the presence of the philadelphia ( ph ) chromosome resulting from the reciprocal translocation t(9;22)(q34;q11 ) . the molecular consequence of this translocation is the generation of the bcr abl fusion gene , which encodes a constitutively active protein tyrosine kinase . the oncogenic protein tyrosine kinase , which is located in the cytoplasm , is responsible for the leukemia phenotype through the constitutive activation of multiple signaling pathways involved in the cell cycle and in adhesion and apoptosis . avascular necrosis of the femoral head ( avnfh ) is not a specific disease . it occurs as a complication or secondary to various causes . these conditions probably lead to impaired blood supply to the femoral head . the diagnosis of avnfh is based on clinical findings and is supported by specific radiological manifestations . we reported a case of a 34-year - old sudanese female with cml who developed avnfh after receiving dasatinib as a second - line therapy . though the mechanism by which dasatinib can cause avascular necrosis ( avn ) is not clear , it can be postulated because of microcirculatory obstruction of the femoral head . to the best of our knowledge and after extensive literature search , this is the first reported case of avnfh induced by dasatinib in a patient with cml .

chronic myelogenous leukemia ( cml ) is a chronic myelo - proliferative disorder with an initially chronic course lasting for 35 years . cml was one of the first diseases in which a specific chromosomal abnormality was identified , a t(9;22)(q34;q11 ) or philadelphia ( ph ) chromosome . avascular necrosis of the femoral head ( avnfh ) occurs as a complicated traumatic or nontraumatic disorder . most cases of avnfh are nontraumatic and occur secondary to excessive corticosteroid use and alcohol abuse.1 other causes include coagulopathies , hemoglobinopathies ( eg , sickle cell disease ) , chronic liver disease , gout , idiopathic hyperlipidemia , metabolic bone disorders , pregnancy , radiation , chemotherapy , smoking , systemic lupus erythematosus , and vasculitis syndromes . intravascular coagulation appears to be the central event associated with nontraumatic avnfh.2 coagulation may occur secondary to extravascular compression ( eg , marrow fat enlargement ) , vessel wall injury ( eg , chemotherapy , radiation ) , or a thromboembolic event ( eg , fat emboli ) . in addition , ischemic insult to the femoral head may result in subchondral bone infarction . in this situation , weakened and unrepaired necrotic bony trabeculae fail under a compressive load , leading to subchondral collapse ( eg , crescent sign ) , and ultimately , articular collapse.3 traumatic causes of femoral head avascular necrosis ( avn ) include femoral neck fractures , hip dislocation , and slipped capital femoral epiphysis.4 avnfh can be presenting manifestation for a patient with cml . we did literature review and came with number of cases reporting avnfh as an initial presentation of cml , illustrated in table 1 . avnfh has been reported as an initial presentation in few cases with cml by many authors ( table 1 ) . a 34-year - old sudanese female with the diagnosis of cml was started on imatinib as a first - line therapy , but she failed the first - line therapy as per european leukemianet guidelines 2010 . she was referred to the hematology service at the national center for cancer care & research ( ncccr ) for further evaluation . her clinical examination was unremarkable , and her work - up was repeated , including a complete blood count , cytogenetics , and bcr / abl by pcr plain radiograph before treatment was available and she was reported as normal . her work - up revealed a chronic phase of cml in failing first - line therapy . she was started on dasatinib 100 mg po once daily as a second - line therapy with which she achieved chr , ccyr , and mmr at 18 months . after 18 months on therapy with dasatinib , she presented with severe pain in her left groin with limping . her peripheral smear reported as normal , and her disease revaluated at the molecular level and showed major molecular response . radiological evaluation , including pelvic radiography ( see fig . 1a and b ) and magnetic resonance imaging ( mri ) showed grade 34 avnfh ( see fig . this is because of the limited number of reports and absence of prospective studies evaluating this issue in this situation . in addition , whether the disease itself and/or the treatment encourage the development of avnfh is not known . post - contrast material - enhanced mri with its inherent high spatial resolution capabilities is considered an excellent diagnostic tool for detecting and staging of femoral head avn . it is considered the preferred method for diagnosis of occult avn , since it is more sensitive than bone scan or plain films . owing to the high incidence of bilateral avn , mri may pick up avn in opposite asymptomatic hip . an avn lesion is typically a well - demarcated epiphyseal area of altered and variable signal intensity ( figs . 1 and 2 ) . in its early stages , t2-weighted images and stir short tau - inversion recovery can help in detection of necrotic tissue in some unusual avn lesions that mostly showed ill - delimited edema - like marrow changes.11,12 mri has 90100% sensitivity for symptomatic disease . in some cases , contrast - enhanced mris may increase diagnostic confidence by showing homogeneous hypervascularization in bone marrow edema lesions and by depicting hypovascular marrow areas in avn lesions . also the mri can discriminate between the avn and transient marrow edema.11,13 sequential and follow - up mri is considered valuable in the assessment of equivocal femoral head lesions , especially in its early stages where the findings are usually trivial.12,13 treatment historically has passed through two eras . the era of interferon : there is a limited data that interferon alfa 2a can cause avnfh . there are no reports in the literature about avnfh with other therapeutic uses of interferon alfa . therefore , the occurrence of avnfh in patients with cml on interferon treatment may be the result of an interaction between cml and interferon alfa therapy . interferon alfa can inhibit angiogenesis , which may cause avn , and the stress of weight bearing may make the femoral head , particularly , vulnerable.14 the second era for cml treatment started with using tyrosine kinase inhibitor ( tki ) table 3 . few cases of avnfh have been reported at disease presentation ( cml ) as well as with the use of first - generation tkis imatinib ( glivec ) . though the mechanism by which dasatinib can cause avn is not clear , it can be postulated because of micro - circulatory obstruction of the femoral head . the above mentioned review of literature states that six patients with cml presented with avnfh as the initial presentation prior to any therapy , five in the era of interferon and two in the era with tkis , and one with imatinib and the other with dasatinib treatment . there are two issues to be considered : either the condition is rare or there is underreporting of this side effect . observational studies with proper reporting are required to accurately measure the incidence of this complication , which could significantly affect patients safety and quality of life .

in the diagnostic work - up of hereditary non - polyposis colorectal cancer ( hnpcc , lynch syndrome ) , high - risk patients can be identified using information from the family history on cancer ( amsterdam criteria and bethesda guidelines ) . to investigate to what extent the medical specialists apply these criteria to patients with colorectal carcinoma and a suspicion of hnpcc , we collected information on diagnostic work - up of 224 patients of seven hospitals in the region of the comprehensive cancer centre west in leiden , the netherlands . these patients were diagnosed with colorectal cancer between 1999 and 2001 and satisfied at least one of the bethesda guidelines . a complete family history was recorded for 38 of the 244 patients ( 16% ) . patients with a complete family history were more likely to be referred to the clinical genetic centre than those with an incomplete or absent family history ( 53% vs. 13% and 4% , respectively ; p < 0.0001 ) , and more likely to be analyzed for microsatellite instability ( msi ) , which is a characteristic of hnpcc ( 34% vs. 6% and 1% , respectively ; p < 0.0001 ) . we conclude that the family history is neglected in the majority of patients with colorectal cancer and msi - analysis is only performed in a small proportion of the patients that meet the guidelines for this analysis .

colorectal cancer ( crc ) is one of the most common forms of cancer in western society . every year 9,500 patients in the netherlands are diagnosed with the disease and almost half of them die from it ( dutch cancer registry , 2002 ) . it is estimated that in around 20% of the patients with a colorectal tumour genetic factors play a role in the aetiology . about 15% of the patients with crc are thought to have hereditary non - polyposis colorectal cancer ( hnpcc , lynch syndrome ) ; a dominant hereditary disease , which is caused by a defect in one of the dna - mismatch repair ( mmr ) genes . the most important clinical characteristics of crc associated with hnpcc , are the relatively young age at which patients are diagnosed with the disease ( average < 45 years old ) and the proximal localisation of the tumour in the colon . besides an increased risk of developing a tumour in the colon , there is an increased risk of developing a tumour elsewhere in the body , especially in the endometrium ( lifetime risk : 50% ) , the small intestines , the ovaries , the brain , the urinary tract , the biliary tract and the development of a keratoacanthoma or a carcinoma of the sebaceous glands of the skin . the identification of patients with hereditary colorectal carcinoma is of great importance for the patient , because the treatment and follow - up of the tumour differ from those with non - hereditary colorectal carcinoma . furthermore , the identification of these patients is important , because it offers efficient manners for the prevention of colorectal carcinoma and other forms of cancer for the patient himself as well as his family . it has been shown that a colonoscopy every three years can lead to a decline in mortality of at least 65% . the genetic defect in hnpcc another method to select families for mutation analysis of the mmr genes is analysis of errors in repetitive dna - sequences , i.e. , micro satellite instability ( msi ) . msi is found in around 15% of the non - selected crc and in more than 95% of the colorectal tumours associated with hnpcc . in 1996 these so - called bethesda - guidelines describe practically all situations , where there is a suspicion of hnpcc . table 1guidelines for the performance of msi - analysis of colorectal tumour revised bethesda - guidelinesa person with colorectal carcinoma diagnosed at age 50a person with colorectal carcinoma and msi - associated pathology<60 yearsa person with colorectal carcinoma and a hnpcc associated tumoura person with colorectal carcinoma and a first degree relative with a colorectal or hnpcc associated tumour ; at least one of the tumours is diagnosed before the age of 50three relatives diagnosed with colorectal carcinoma or a hnpcc associated tumour , diagnosed at any age ; one patient needs to be a first degree relative of the other twothe presence of tumor - infiltrating lymphocytes , so called , " crohn 's like lymfocyte reaction " , mucinous or signet ring cell carcinoma differentiation or medullary growth patterncarcinoma of the endometrial tissue , stomach , small intestines , pancreatic gland , biliary tract , urinary tract , ovaries , brain , keratoacanthoma and carcinoma of the sebaceous glands guidelines for the performance of msi - analysis of colorectal tumour the presence of tumor - infiltrating lymphocytes , so called , " crohn 's like lymfocyte reaction " , mucinous or signet ring cell carcinoma differentiation or medullary growth pattern carcinoma of the endometrial tissue , stomach , small intestines , pancreatic gland , biliary tract , urinary tract , ovaries , brain , keratoacanthoma and carcinoma of the sebaceous glands before the discovery of the mmr - genes , the most common approach in the diagnostic work - up for hnpcc was to use the amsterdam criteria. these criteria are met if there are , within one family , three individuals with a colorectal ( or another hnpcc - associated kind of ) tumour , of whom one person is a first degree family member of the other two and at least one carcinoma is diagnosed before the age of fifty . to evaluate the amsterdam criteria in patients with crc , a complete history on cancer in the patient s family has to be obtained . until now it is not known whether an adequate family history is taken of all patients with a colorectal carcinoma . we also do not know to what extent medical specialists use the above mentioned clinical bethesda guidelines and if the tumours of all the patients who match the criteria are tested for msi . the objective of the present study was to answer these questions using data of the cancer registry of the comprehensive cancer centre west ( cccw ) in leiden , the netherlands . we selected patients who were diagnosed with a primary and invasive colorectal tumour in the period 19992001 from the cancer registry ( cr ) of the comprehensive cancer centre west ( cccw ) in the netherlands . the patients had to satisfy one of the following two bethesda guidelines : the patient had to have more than one tumour , i.e. , one colorectal carcinoma and a second one ( colorectal cancer or another hnpcc - associated kind of tumour ) , or the patient had to be fifty years or younger at diagnosis . the selected patients were considered to have an indication for the performance of msi - analysis and/or referral to the clinical genetic centre ( cgc ) . patients with a carcinoma in situ or a carcinod of the appendix were not included in the analysis . between 1999 and 2001 , 434 patients who complied with the above mentioned criteria were diagnosed with crc in one of the twelve hospitals in the cccw - region . seven hospitals gave permission for the collection of information concerning family history , msi - analysis and referral to the cgc . the family history was considered complete if the medical records reported on cancer in the family , and if so , information about the age at the time of the diagnosis , the type of cancer and the occurrence of cancer within first - degree and second - degree family members . 120 patients had multiple tumours , 109 patients were fifty years or younger at the time of diagnosis , and 15 patients had both characteristics . for comparisons between patients with multiple tumours and patients who were young at diagnosis , those with both characteristics were allocated to the multivariate logistic regression analysis was used to study whether the presence of a complete family history or referral to a cgc could be explained by age , sex , inclusion criterion ( multiple tumours or young age at diagnosis ) , hospital or type of medical specialist . the study group consisted of 244 persons , who complied with one of the bethesda guidelines and therefore were considered to be referred for msi - analysis and/or genetic counselling . the male : female ratio was 49:51 and did not differ between the groups selected on the basis of multiple tumours or age 50 years at diagnosis . a complete family history was recorded in the medical records of 38 ( 16% ) of the 244 patients . for 136 patients ( 55% ) limited information on the family history was available , and for 70 ( 29% ) patients no information on the family history of the 38 patients with a complete family history , 20 ( 53% ) were referred to the cgc . this percentage was higher than that of patients with an incomplete family history ( 13% ) and that of patients without any information on family history ( 4% ) ( p < 0.0001 , table 2 ) . msi - analysis was performed more often in the patients with a complete family history : 34% of patients with a complete family history compared to 6% of patients with an incomplete family history and 1% of patients without any family history ( p < 0.0001 ) ( table 2 ) . presence of a complete family history and the performance of msi - analysis were not associated with age , sex , inclusion criterion ( multiple tumours or young age at diagnosis ) , hospital or type of medical specialist ( multivariate logistic regression analysis ; data not shown ) . table 2diagnostic work - up for hnpcc in 244 patients with colorectal cancer , by completeness of the family history as reported in the medical recordsdiagnostic workupfamily history complete ( n = 38)family history incomplete ( n = 136)family history absent ( n = 70)referred to cgc20 ( 53%)17 ( 13%)3 ( 4%)msi - analysis performed13 ( 34%)8 ( 6%)1 ( 1%)results of msi - analysis3 msi , 10 stable7 stable , 1 unknown1 stablediagnosis of hnpcc6 ( 16%)3 ( 2%)1 ( 1% ) diagnostic work - up for hnpcc in 244 patients with colorectal cancer , by completeness of the family history as reported in the medical records we used the bethesda - guidelines to select a group of patients with a suspicion of hnpcc . these patients were diagnosed with colorectal cancer between 1999 and 2001 , a period during which msi - analysis and the bethesda guidelines were already available . therefore we expected that for these patients , physicians would have examined and reported their patients family history and that msi - analysis would have been performed . in our study group , however , the family history of the patients diagnosed with colorectal carcinoma was not sufficiently examined and reported in the medical records . for this reason , we believe the bethesda - guidelines were not sufficiently applied by the physicians . as a consequence , msi - analysis was performed on a small proportion of the tumours . more patients with a complete family history in their medical records were referred by their physicians to the cgc than patients without such a family history . we expect that in a low - risk population , i.e. , patients with colorectal cancer who do not meet the bethesda guidelines , these results would be even more dramatic . on the one hand we collected our data using medical records from various medical specialties , while the treating physician will not have this overview in practice . on the other hand , it is possible that when a physician examined a family history and none of the family members was diagnosed with cancer , he did not report it in the medical records . in this case , the family history was considered as absent , although it in fact was examined . nevertheless we expect that if msi - analysis was performed or the patient was referred to the cgc , this would have certainly been reported . we found that the attention for hnpcc in the diagnostic workup for crc differed widely . for the seven participating hospitals , the proportion of patients with a reported family history on cancer ranged from 38% to 91% . for these reasons , we can not generalise our results for the whole cccw - region . nevertheless , we conclude that the family history appears to be neglected in the majority of patients with colorectal cancer in our study period , and that msi - analysis was only performed in a small proportion of the patients that meet the guidelines for this analysis . possibly , the attention for identification of patients with hnpcc has increased in more recent years . our findings underscore the importance of implementation of family history and bethesda guidelines in the physician education .

background . bronchopulmonary dysplasia ( bpd ) is the most common chronic lung disease of premature birth , characterized by impaired alveolar development and inflammation . pathomechanisms contributing to bpd are poorly understood . however , it is assumed that genetic factors predispose to bpd and other pulmonary diseases of preterm neonates , such as neonatal respiratory distress syndrome ( rds ) . for association studies , genes upregulated during alveolarization are major candidates for genetic analysis , for example , matrix metalloproteinases ( mmps ) and fibroblast growth factors ( fgfs ) and their receptors ( fgfr ) . objective . determining genetic risk variants in a caucasian population of premature neonates with bpd and rds . methods . we genotyped 27 polymorphisms within 14 candidate genes via restriction fragment length polymorphism ( rflp ) : mmp-1 , -2 , -9 , and -12 , -16 , fgf receptors 2 and 4 , fgf-2 , -3 , -4 , -7 , and -18 , signal - regulatory protein ( sirpa ) and thyroid transcription factor-1 ( ttf-1 ) . results . five single nucleotide polymorphisms ( snps ) in mmp-9 , mmp-12 , fgfr-4 , fgf-3 , and fgf-7 are associated ( p < 0.05 ) with rds , defined as surfactant application within the first 24 hours after birth . one of them , in fgfr-4 ( rs1966265 ) , is associated with both rds ( p = 0.003 ) and bpd ( p = 0.023 ) . conclusion . rs1966265 in fgf receptor 4 is a possible genetic key variant in alveolar diseases of preterm newborns .

great progress has been made in neonatal care over the last few decades , reflected by improving survival rates and clinical outcomes of preterm infants . despite these advances , 45 years after its first description , bronchopulmonary dysplasia ( bpd ) remains a major complication of premature birth , causing ongoing morbidity and mortality : it is the most common neonatal chronic lung disease , affecting around 25% to 35% of vlbw neonates ( very low birth weight , < 1500 g ) , and is associated with increased risk for rehospitalization [ 3 , 4 ] , cognitive delay , and neurosensory deficits . initially described by northway et al . in 1967 , the old bpd mainly affected modestly premature newborns suffering from respiratory distress and therefore mechanically ventilated with high levels of supplemental oxygen . with the introduction of surfactant treatment , prenatal maternal use of glucocorticoids , improved nutrition , and ventilator strategies the clinical course and pathology of bpd have changed considerably . unlike the original description , today 's new bpd is mainly regarded as a disruption of distal lung growth [ 6 , 7 ] . thus , influenced by both genetic susceptibility [ 8 , 9 ] and environmental factors on the immature lung , the pathophysiology is characterized by inflammation , abnormal microvascularization , and impaired alveolarization . alveolar formation of the primitive saccules is a complex process of epithelial morphogenesis , capillary growth , and coordinated extracellular matrix ( ecm ) remodelling . at this , fibroblast growth factor ( fgf ) signalling and matrix metalloproteinase ( mmp ) activity play eminent roles . some mmps are upregulated in inflammatory environment and yet are involved in pulmonary host defense . there is evidence for some mmp isoforms being important determinants for alveolarization , especially mmp-2 , -9 , and -16 : mmp-2 deficient mice showed fewer and larger alveoli with thinner interstitial tissue . hadchouel et al . demonstrated an increase of mmp-16 activity during the alveolar stage and moreover found two snps within the mmp-16 gene being associated with lower tracheal mmp-2 and -16 activity and to protect from bpd . prospecting further potential biomarkers for bpd , also mmp-9 shows some promise ; for example , harijith et al . highlighted a mmp-9-dependent lung injury pathway in an ifn-mediated animal model of bpd . mice with a partial mmp-9 deficiency showed a reversal of ifn-induced lung injury during hyperoxia . mmps , particularly mmp-2 and -9 , activate fibroblast growth factors ( fgfs ) by cleavage in the ecm , especially during angiogenesis . in turn activated fgfs upregulate mmp expression . fgfs are secreted glycoproteins involved in interactions between epithelium and mesenchyme regulating cell migration and proliferation in embryonic development , especially in fetal pulmogenesis [ 16 , 17 ] . their signalling depends on membrane - located receptors ( fgfrs ) with a tyrosine kinase domain , encoded by four different genes ( fgfr 14 ) [ 1820 ] . they are all translated in developing lungs and are suggested to play major roles in modifying distal lung patterns during alveolarization ; for example , fgfr-3-fgfr-4 double - knockout mice show no alveolarization . it has been assumed that heritable determinants contribute significantly to both bpd [ 8 , 23 ] and rds . on this account , we were interested in identifying genetic risk factors in a caucasian population of premature newborn with bpd and rds . we genotyped 27 polymorphisms within fourteen candidate genes for bpd : mmp-1 , -2 , -9 , -12 , and -16 , fgf receptors 2 and 4 , fgf-2 , -3 , -4 , -7 , and -18 , signal - regulatory protein ( sirpa ) , and thyroid transcription factor-1 ( ttf-1 ) . we also included sirpa because of the known effect on surfactant proteins and inhibition of macrophages , as well as ttf-1 due to its effect on lung differentiation . we recruited preterm neonates ( 28 weeks of gestation ) born between january 1996 and september 2010 at the centre for pediatrics and adolescent medicine , university hospital freiburg , germany . twins and siblings were excluded from the study as were children with chromosomal aberrations , congenital heart defects , or other major congenital malformations . dna was collected by buccal swabs or by routine blood sampling , between 2 weeks up to 2 years of age . this included gestational week , number of days with supplemental oxygen , need of mechanical ventilation and positive airway pressure , and need of surfactant therapy . as described previously , the subdivision of our bpd study population was based on the analysis by lavoie et al . about the heritability of bpd according to the consensus defined by the national institute of health : the bpd population included all infants with moderate and severe bpd , that is , supplemental oxygen for at least 28 days plus need of oxygen and/or positive pressure at 36 weeks of gestation , whereas the control population consisted of all preterm neonates with no or mild bpd . recruiting neonates for the rds population was targeted on severe cases of respiratory distress by including only newborns depending on surfactant within the first 24 hours after birth ( see supplementary material available online at http://dx.doi.org/10.1155/2013/932356 ) . at our neonatal intensive care unit ( nicu ) the following approach has been applied regarding the treatment with surfactant : avoiding of intubation independent of the gestational week . therefore , even very premature infants are only intubated if they show failure of ventilation and/or need of supplemental oxygen above 40% . once they required intubation during the immediate postnatal period , they receive surfactant within 2 hours . we included a minority of polymorphisms that already had been tested for other pathologies ( rs1799750 in mmp1 and rs2276109 and rs652438 in mmp12 ) . for pcr reactions , genomic dna was initially denatured at 94c for 5 minutes and underwent 3540 cycles of denaturation ( 94c for 30 seconds ) , annealing ( 1 minute , corresponding temperatures displayed in table 2 ) , extension reaction ( 72c for 1 minute ) , and a final extension step at 72c for 8 minutes . in table 2 some primers contain intended single nucleotide mismatches ( mutagenic primers ) to create sites for restriction enzymes . accuracy of the rflp was confirmed by sequencing via dideoxy chain termination method , respectively , three controls ( homozygous wildtype , heterozygous , and homozygous mutation ) for each polymorphism using the big dye terminator cycle sequencing kit on an abi 310 sequencer ( applied biosystems ) . genotyping data of our case - control populations were analysed by using armitage 's trend test ( att ) for possible association with bpd and rds as specified previously . moreover att was used to calculate hardy weinberg equilibrium ( hwe ) for each polymorphism . the collection of blood / buccal swabs and the experimental procedures were approved by the ethical committee of the university of freiburg . parents were given written and verbal information about the study and a statement of informed consent was signed by the parents of all enrolled children . the results of the 27 studied polymorphisms ( table 1 ) for association with bronchopulmonary dysplasia and neonatal respiratory distress are specified in table 3 ( bpd ) and table 4 ( rds ) . among the 11 genotyped polymorphisms in different mmp genes ( see table 1 ) there was no bpd - associated polymorphism ( table 3 ) but two polymorphisms associated ( p < 0.05 ) with rds ( rs20544 in mmp-9 : p = 0.033 ; rs652438 in mmp-12 : p = 0.047 , see table 4 ) . both snps show no significant deviation from hardy - weinberg equilibrium , neither in the control nor in the case population . analysis of rs20544 ( c / t ) identifies the t allele as protective against respiratory distress . for the genotyping results of the amino acid substitution rs652438 ( a / g , asn357ser ) the complete absence of the g / g homozygous genotype in the respiratory distress case population must be taken in account . the other mmp - snps showed no association , inclusively rs2664352 in mmp16 , that had been associated with protection from bpd . the fgfr-4 snp rs1966265 , located in the exon region and causing an amino acid substitution of isoleucine ( ile ) for valine ( val ) is associated with both bpd ( p = 0.023 ) and rds ( p = 0.003 ) . here the a / a genotype ( ile ) could be identified as protective allele variant against our studied lung diseases . the association results from significant differences in allele frequencies : in both bpd and rds analysis the g allele is more frequent in the disease populations ( see tables 3 and 4 ) . the other snps in the fgfr genes showed no association with neither bpd nor respiratory distress . whereas no association could be detected between the eight fgf - snps and bpd , rs10796856 in fgf-3 and rs4316697 in fgf-7 showed associations with rds . correspondent p values are p = 0.036 ( rs10796856 ) and p = 0.044 ( rs4316697 ) , and no deviations from hardy - weinberg equilibrium were detected ( see table 4 ) . the four snps in sirpa and ttf-1 showed no association with neither bpd nor rds . analysis of ttf-1 rs999460 unfolds deviation from hardy - weinberg equilibrium in both case and one control populations in our caucasian population ( see tables 3 and 4 ) . the aim of this study has been to identify genetic risk factors in an ethnically homogenous caucasian population . genetic contribution to bpd is suggested on the basis of twin studies demonstrating that at least half of the susceptibility is hereditary [ 8 , 9 , 23 ] . additionally , lavoie et al . could differentiate in their study that mild bpd ( according to the national institute of child health and human development consensus definition ) had been mainly attributable to shared environmental factors whereas moderate or severe bpd had been attributable to genetic influence . following these findings , we defined our control population as neonates with no bpd or mild bpd , whereas our bpd population included neonates with moderate or severe bpd . furthermore , we recruited only preterm neonates 28 weeks of gestational age for the bpd population to avoid false associations based on the fact that bpd hardly develops in newborn older than 30 weeks of gestational age . in contrast to bpd , the results of twin studies on rds susceptibility showed mostly contradictory results [ 24 , 3235 ] . a twin study by levit et al . with 332 twin pairs of a heterogeneous population has been the first one to include and assess the influence of several independent covariates , revealing that 50% of the variance to rds susceptibility is hereditary . given these lines of evidence for genetic contribution , we have chosen the candidate - gene approach for our association study based on the hypothesis that genes fundamental in lung organogenesis and alveolar remodelling , that is , mmp and fgf , determine susceptibility to bpd and rds . known genetic risk factors for rds are mostly allelic polymorphisms of the genes encoding surfactant proteins sp - a1 , sp - a2 , and sp - b . anyhow , other determinants than components of the surfactant system might also affect the liability to rds . genes encoding for growth factors or enzymes that account for alveolarization through proper secondary septation and extracellular remodeling might affect the gas - exchange and therefore aggravate respiratory distress at birth . supposed genetic risk factors for bpd are mostly genes encoding components of innate immunity and antigen - presentation , cytokines , antioxidant defences , and angiogenic growth factors such as : mannose - binding lectin ( mbl2 ) , tumor necrosis factor - alpha ( tnf- ) [ 28 , 38 ] , human leucocyte antigen ( hla)-a , -b , and -c alleles , glutathione - s - transferase - p1 , and vascular endothelial growth factor ( vegf ) . some years ago , two mmp-16 gene polymorphisms were demonstrated to protect from bpd and moreover to be associated with lower tracheal mmp-2 and -16 levels . matrix metalloproteinases are a family of zinc - dependent endopeptidases , and they degrade extracellular components and play a crucial role in lung development , especially during alveolarization . particularly mmp-2 and -9 ( so - called gelatinases a and b ) seem to be relevant in extracellular remodeling and even pulmonary host defense . they degrade type iv collagen , fibronectin , elastin , and denatured collagen ( gelatin ) . mmp-2 deficient mice show an abnormal saccular development with larger and simplified alveoles . in line with this finding , newborns developing bpd showed low mmp-2 tracheal levels at birth [ 41 , 42 ] . recently , mmp-9 could be identified as a pathogenic key mediator in a murine model of bpd . on the other hand , increased tracheal levels of mmp-9 early after birth have been associated with resolving rds , suggesting that increase in mmp-9-activity is a physiologic repair response . demonstrated that increased mmp-9 activity in neonatal lungs early after birth correlated with resolving respiratory distress syndrome , demonstrating a likely role of mmp-9 in pulmonary host defense . in our study we identified an snp ( rs20544 ) in the mmp-9 gene to be associated ( p = 0.033 ) with rds , but not bpd . respiratory distress syndrome has been defined as need of surfactant ( see supplementary material ) . on one hand , ethnically homogenous populations like our caucasian population are favourable to detect possible pathogenetic determinants , but one must bear in mind that the size of our rds population is limited and the total numbers of neonates studied for each polymorphism vary slightly according to the recruiting time point . furthermore , association studies on rds are prone to confounding factors . other pulmonary conditions such as a transient tachypnea provoked by wet lung syndrome or pulmonary infection might mimic respiratory distress syndrome caused by surfactant deficiency and thereby hamper the results of our study . in our study , we included mmp-16 polymorphisms that had been associated with bpd in a french population ( rs2664352 ) . in our population rs2664352 up to now , the role of fgf3 has been mainly studied in cancer diseases , that is , lung cancer , but its exact role in pulmogenesis remains elusive . there is evidence for fgf-3 upregulation to be associated with alveolar type 2 cell hyperplasia and downregulation to be associated with an excessive recruitment of free alveolar macrophages which might lead to symptoms of respiratory distress . furthermore it has been shown that fgf-3 stimulates the secretion of mmp-2 and -9 propeptides in vitro . fgfr-4 polymorphism rs1966265 showed association with both respiratory distress ( p = 0.003 ) and bronchopulmonary dysplasia ( p = 0.023 ) . the a / a genotype ( encoding for isoleucin instead of valine ) has been protective in our association study . the exact - test showed no deviation from hardy weinberg equilibrium for this snp in both case and control populations , suggesting that the association does not result from population admixture or genotyping errors . fgfr-1 to fgfr-4 are expressed in the lung and fgfr-3 and -4 signalling , in particular , appears to be fundamental in alveolar formation . weinstein et al . demonstrated that mice deficient in both fgfr-3 and -4 show a completely blocked alveolarization and fail to show any formation of secondary septae , whereas solely fgfr-4(/ ) animals exhibit no significant abnormalities , revealing a cooperative effect of fgfr-3 and -4 in lung development . hyperoxia - exposed ( fio2 0.85 ) mice show a bpd - like lung pattern of enlarged airspaces and furthermore a reduced expression of fgfr-3 and -4 , suggesting a pathogenic role in arrested lung development . replicated these results in fgfr-3 and -4 deficient mice and demonstrated in addition that fgfr-3/-4 signaling contributes to excessive elastin production and its alveolar accumulation , which is another typical feature of bpd . but these abnormalities have not been due to fibroblast defects but due to increased expression of paracrine factors of alveolar type 2 cell ( at2 ) . if a reduction in fgfr-3 and -4 expression affects distal lung development , a functionally significant polymorphism within the correspondent gene possibly alters the susceptibility to alveolar disease such as bpd and rds . showed that there is a peak of fgfr-4 expression at the day of birth , when respiratory distress syndrome occurs . false - positive results can only be excluded by replications in other study populations . in conclusion , we describe five snps in mmp-9 , mmp-12 , fgfr-4 , fgf-3 , and fgf-7 that are associated ( p < 0.05 ) in our caucasian population with respiratory distress syndrome of the newborn , defined as surfactant application within the first 24 hours after birth . among these polymorphisms one polymorphism in fgfr-4 ( rs1966265 ) is additionally associated with bronchopulmonary dysplasia , demonstrating its possible role in the pathogenesis of newborn lung diseases on grounds of pulmonal immaturity .

background and aims : the aim was to evaluate efficacy of optic nerve sheath diameter ( onsd ) by ultrasound as a noninvasive method for detecting raised intracranial pressure ( icp ) in intensive care unit , to compare with computed tomography / magnetic resonance imaging ( mri ) findings of raised icp and to prognosticate onsd value with treatment.materials and methods : we conducted a prospective , observational study on 101 adults by including 41 healthy individuals in group a as control and 60 patients in group b admitted with fever , headache , vomiting , and altered sensorium . we examined them in supine position using 10 mhz linear array probe on closed eyelid . onsd was measured 3 mm behind the globe in each eye . a mean binocular onsd > 4.6 mm in female and 4.8 mm in male was considered abnormal . midline shift , edema , effacement or onsd > 5.0 mm on t2 mri suggestive of elevated icp was used to evaluate onsd accuracy.results:group a mean onsd was 4.6 mm in females and 4.8 mm in males . group b mean onsd for 17 females was 5.103 0.6221 mm ( p = 0.002 ) and for 43 males 5.081 0.5799 mm ( p = 0.032 ) . radiological sign of raised icp was confirmed in 35 patients ( females = 11 and males = 24 ) with high onsd value . sensitivity of detecting raised icp by onsd was 84.6% in females and 75% in males while specificity was 100% in both genders . out of 25 patients without radiological signs of raised icp 10 patients showed high onsd ( females = 4.735 mm and males = 4.907 mm ) . onsd was well prognosticated with treatment modalities.conclusion:bedside ocular ultrasonography for measuring onsd can be used an early test for diagnosing raised icp as it is a noninvasive , cost effective bedside test , which can be repeated for re - evaluation .

raised intracranial pressure ( icp ) is usually associated with increased morbidity , mortality , and poor neurological outcomes . the etiology could be varied viz stroke , liver failure , meningitis , meningoencephalitis , metabolic encephalopathy and postresuscitation syndrome . it is associated with complications such as infection , bleeding and being expensive . regular assessment and comparison by computed tomography ( ct)/magnetic resonance imaging ( mri ) in these critically ill - patients the optic nerve , as a part of the central nervous system , is surrounded by a subarachnoid space and experiences the same pressure change as the intracranial compartment . the intraorbital part of the sheath , and particularly its retrobulbar segment , can distend when icp is elevated . the use of bedside ocular ultrasonography ( usg ) in measuring optic nerve sheath diameter ( onsd ) can be a useful method for detecting raised icp . it has the advantage of being a noninvasive , portable , easily performed at bedside in minimum time . it can be repeated for re - evaluation without risk of radiation . keeping this in view , we conducted the bedside study of a noninvasive measurement of onsd as predictor for detecting raised icp . we conducted a prospective observational study on 101 adult individuals after institutional review board 's permission over a period between may 2013 and august 2013 . they were divided into two groups a and b. group a were , 41 healthy controls of which 20 were female and 21 male . a total of 60 patients were admitted during this period with symptoms of fever , headache , vomiting and altered sensorium with possibility of elevated icp , were included in group b. they were 17 female and 43 male . all these patients were examined in the supine position using a 10 mhz phased linear array probe on the closed eyelids [ figure 1 ] . the structures of the eye were visualized to align the optic nerve directly opposite the probe , with the onsd width perpendicular to the vertical axis of the scanning plane . a single onsd was measured 3.00 mm behind the globe [ figure 2 ] in both the eyes . the onsd measurements were obtained averaging three readings from each eye to create a binocular onsd measurement . occular sonography : high frequency 10 mhz linear array probe placed on the gel on the closed eyelid the optic nerve sheath diameter ( onsd ) measurement : optic nerve appears homogeneous with low internal reflectivity compared with the high reflectivity of the nerve sheath . onsd measured 3 mm behind the globe using an electronic caliper with an angle perpendicular to the eye ball the individuals in the control group were between 18 and 40 years . in these controls a mean binocular onsd was 4.6 mm and 4.8 mm in females and males respectively . the measurements above 4.6 mm and 4.8 mm in females and males were considered to have increased icp . imaging of the head ct / mri was done as per requirement in group b patients only . the finding of ct / mri was reported by the on - site radiologist , and they were correlated with bedside onsd measurement . the patient 's imaging result was considered to be positive for raised icp if the radiologist 's impression described findings , suggestive of elevated icp such as significant cerebral edema , midline shift , mass effect , effacement of sulci , collapse of ventricles , compression of cisterns and onsd > 5.0 mm on t2 mri . the waiver of the individual consent was requested as the intervention was completely harmless , nonchargeable to the patient and no patient identification was used . patients with a history of optic neuritis , arachnoid cyst of the optic nerve , high myopic , optic nerve trauma , and anterior orbital or cavernous sinus mass are excluded from the study . the variables used were age , sex , diagnosis , heart rate , blood pressure , respiratory rate , oxygen saturation , temperature , glasgow coma scale ( gcs ) , onsd , signs of raised icp on imaging , serum osmolarity , serum creatinine , mannitol , and management . statistically assessed the observed differences among various onsd groups in the distribution of categorical variables , the pearson or fisher exact test of association was used . statistics to test baseline differences between the study group and controls was performed with the mann - whitney u - test . statistically assessed the observed differences among various onsd groups in the distribution of categorical variables , the pearson or fisher exact test of association was used . statistics to test baseline differences between the study group and controls was performed with the mann - whitney u - test . statistically assessed the observed differences among various onsd groups in the distribution of categorical variables , the pearson or fisher exact test of association was used . statistics to test baseline differences between the study group and controls was performed with the mann - whitney u - test . a were 41 ( females = 20 and males = 21 ) as a control . in group the mean onsd of control and study group for female was 4.627 0.09 mm and 5.103 0.62 mm and for male was 4.8 0.10 mm and 5.081 0.58 mm respectively . the mean age of the control and study group was 27.44 3.31 and 56.15 18.86 years respectively . demographic profile of the study patient out of 60 patients that were admitted , 35 cases showed raised icp on imaging and their onsd 5.43 0.53 mm . the other 25 patients did not show a raised icp on imaging and onsd was 4.61 0.19 mm . it showed a significant difference between the two sets of patients with a p < 0.001 . however , 10 out of their 25 showed a rise in onsd for a female 4.735 mm > 4.6 mm and male 4.907 mm > 4.8 mm which required reduction of icp . the mean gcs for raised icp and normal icp on imaging was 10.5 0.12 and 12.04 2.11 respectively . the mean temperature 99.31 0.75f and 98.89 0.55f respectively for raised icp and normal icp . it showed statistically significant as p value for gcs and temperature was 0.04 ( < 0.05 ) and 0.02 ( < 0.05 ) respectively . other variables were also analyzed and did not show any statistically significant difference as shown in table 2 . association of icp with clinical profile receiver operator characteristic ( roc ) curve ( area under the curve [ auc ] ) for mean onsd 4.716 mm is 98.6% ( 95% ci : 96.5 - 100% ) with sensitivity of 77.8% and specificity of 100% . for female roc curve ( auc ) for onsd > 4.6 mm is 100% ( 95% ci : 100% ) , with sensitivity of 84.6% and specificity of 100% whereas for male roc curve ( auc ) for onsd > 4.8 mm is 97.4% ( 95% ci : 93.5 - 100% ) with sensitivity of 75.0% and specificity of 100% as shown in figures 3 and 4 . receiving operating characteristic curve for detecting raised intracranial pressure by ultrasonography optic nerve sheath diameter for female receiving operating characteristic curve for detecting raised intracranial pressure by ultrasonography optic nerve sheath diameter for male all these patients received treatment with mannitol , antiepileptics , antiplatelet , antibiotics , and antiviral depending on cerebrospinal fluid ( csf ) analysis . the trend of onsd in patients with raised icp was recorded from day 1 to day 4 . in the female population onsd on day 1 was 5.405 0.5729 mm and by day 4 it decreased to 4.868 0.4417 mm . similarly in male onsd on day1 was 5.442 0.5233 mm and by day 4 it decreased to 5.065 0.3730 mm . in 10 cases that did not show raised icp on imaging but had increased onsd on day 1 for female 4.735 0.064 mm and male 4.907 0.054 mm . reduction of icp was noted on 2 day as onsd decreased to 4.630 0.071 mm in female and to 4.823 0.038 mm in male . trend of onsd with treatment trend of onsd with treatment about 35 patients had raised icp on imaging and onsd of 5.430 0.5311 mm , 21 cases diagnosed as infective ( meningitis , meningoencephalities ) , 12 cases were cerebrovascular accident ( stroke , intracranial hemorrhage ) , and 2 cases were metabolic encephalopathy . whereas out of 25 cases that did not show raised icp radiologically 2 cases were infective ( meningitis , meningoencephalities ) , 18 cases were cerebrovascular accident ( stroke , intracranial hemorrhage ) and 5 cases were metabolic encephalopathy . but 10 cases did not show raised icp radiologically but had mean onsd for two female as 4.735 mm and eight male as 4.907 mm . they were 8 cerebrovascular accident , 1 metabolic encephalopathy , and 1 infective . in the study group out of 60 patients , 28 patients had high onsd and signs of raised icp on imaging received mannitol as definitive treatment . however , three patients who showed only high onsd and did not show raised icp on imaging also received mannitol and they showed a decrease in onsd after supportive therapy . totally 26 patients required intubation and controlled mechanical ventilation with sedation . in the study group , 21 patients had high onsd and signs of raised icp on imaging , but the other five patients had high onsd , but did not show signs of raised icp on imaging . total 5 cases expired , 2 with icp increased , 3 without icp and it did not find any statistical significant . raised icp is , usually , associated with conditions such as stroke , liver failure , meningitis , meningoencephalitis , metabolic encephalopathy , and postresuscitation syndrome . the cranium and the vertebral canal along with the relatively inelastic dura form a rigid container , such that the increase in any of its contents , that is , brain , blood , or csf , will tend to increase the icp . the monro - kelliy doctrine relationship states that small increases in brain volume do not lead to immediate increase in icp due to the ability of the csf to be displaced into the spinal canal , as well as the slight ability to stretch the falx cerebri between the hemispheres and the tentorium between the hemispheres and the cerebellum . however , once the icp reaches around 20 - 25 mmhg , any small increase in brain volume can lead to marked elevations in icp due to failure of intracranial compliance . in stage 1 , there is a minimal increase in icp . in stage 2 , there is a drastic increase in icp as change in volume is > 100 - 200 ml . stage 3 is characterized by a sustained increased icp , with dramatic changes in icp with small changes in volume . as the icp approaches the mean arterial pressure , it becomes more and more difficult to squeeze blood into the intracranial space , leading to widespread reduction in cerebral flow and perfusion , eventually leading to ischemia and brain infarction . because of hypoxia and hyper apnea , patients present with decreased level of consciousness ( loc ) , cheyne - stokes respiration , hyperventilation , sluggish or dilated pupils and widened pulse pressure . generally when there is a rise in icp , common symptoms and signs include headache , vomiting without nausea , and altered loc . the headache is worse on coughing / sneezing / bending , and progressively worsens over time . the optic nerve sheath ( ons ) is anatomically continuous with the dura mater and has a trabeculated arachnoid space through which csf slowly percolates . on ultrasound examination , optic nerve appears homogeneous with low internal reflectivity compared with the high reflectivity of the nerve sheath ; this was utilized by ossoinig when he performed the first ultrasound measurement of the optic nerve using an a - scan technique , and subsequently described standardized a - scanning . using these echography techniques several groups have investigated the relation between the onsd as measured by a - scan and the icp cennamo et al . each demonstrated a positive linear relation between these two variables in neurosurgical patients and in particular , an immediate change in onsd with change in icp . a position 3 mm behind the globe was chosen because the ultrasound contrast is greatest , the results are more reproducible , and anatomically the anterior nerve is most distensible . hansen et al . presented data using a transorbital b - scan approach for the measurement of onsd , this approach allowed them to select a distance behind the globe to consistently measure the nerve , something difficult to attain with a - scan techniques . helmke and hansen demonstrated in cadaver studies that the onsd increased by up to 60% at a distance of 3 mm behind the globe compared with only 35% at 10 mm thus confirming liu and kahn 's observations . furthermore , they went on to show that the optimal experimental scanning position was longitudinal ( axial ) where the least interobserver variability was found although there was no significant difference in measurement by lateral , axial , or transverse projection . in our study , the average onsd in the control group aged between 18 and 40 years was 4.6 mm and 4.8 mm in female and male respectively . it has been proved by dubourg et al . in a systematic review and meta - analysis of a ultrasonographic of onsd of detection of raised icp , a onsd in a adult < 5 mm , pediatrics ( 1 - 15 years ) they also concluded that onsd > 5.00 - 5.70 mm had a raised icp > 20 mm with diagnostic odds ratio of 51 , sensitivity of 90% ( 95% ci : 80 - 95% ) and specificity 85% ( 95% ci : 73 - 93% ) . in a study conducted by dubost et al . to detect the incidence of raised icp in preeclampatic patients , concluded that onsd was 5.4 mm in preeclampatic patients when compared to healthy pregnant women which was 4.5 mm . similar study conducted by rajajee et al . who concluded that bedside measurement of onsd is an accurate noninvasive method to identify icp > 20 mmhg in a heterogeneous group of patients with acute brain injury with onsd > 4.8 mm has greatest accuracy . in 60 admitted patients , 35 cases had raised icp on radiological findings , the onsd for female was 5.405 mm and male was 5.442 mm . about 10 ( 40% ) individuals did not have raised icp on imaging , but their onsd was found to be more than the control for female 4.735 0.064 mm and male 4.907 0.054 mm . we found for mean onsd of 4.716 mm , sensitivity for detecting raised icp was 77.8% ( 95% ci : 83 - 100% ) and specificity 100% . in female sensitivity for raised icp is 84.6% and specificity 100% . in male sensitivity for raised icp was 75.0% and specificity was 100% . beare et al . evaluated ons ultrasound as a noninvasive method of detecting raised icp in african children . they concluded that sensitivity and specificity of detecting raised icp on ct for onsd 4.2 mm was 100% and 86% respectively . conducted a prospective blinded observational study on adult head injury patients in an emergency department . it has been proved by dubourg et al . in a systemic review and meta - analysis of a ultrasonographic measurement of ons diameter of detection of raised icp , that onsd > 5.00 mm had a raised icp > 20 mm , pooled sensitivity 90% ( 95% ci : 80 - 95% ) , specificity 85% ( 95% ci : 73 - 93% ) . the patients with raised icp are 51 times more likely to have a positive onsd . it was also noted that papilledema was not found in the acute situation as it takes hours to days to develop . acute rise in icp can be difficult to diagnose because the symptoms are nonspecific , and direct measurement of icp has the attendant risks of intracranial hemorrhage and infection . in our analysis , 35 patients who had raised icp on ct / mri only temperature and gcs had a significant difference [ table 2 ] . about 35 cases had raised icp on imaging and high onsd on the 1 day of admission and after starting the appropriate treatment onsd was measured subsequently on days 2 , 3 and 4 . we found a significant reduction of onsd for female and male after 72 h of treatment [ table 3a ] . about 25 cases did not have increased icp on imaging . however 10 cases out of 25 had onsd higher than the cutoff value for female 4.735 0.064 mm and male 4.907 0.054 mm [ table 3b ] . after supportive treatment , their onsd on day 2 for female and male was almost returned to normal value . the early detection of raised icp can be very difficult when invasive devices are not available . clinical signs of raised icp such as headache , vomiting and drowsiness are not specific and often difficult to interpret . in sedated patients , clinical signs of raised icp bedside measurement of onsd is a useful test to identify raised icp , being noninvasive , can be repeated multiple times , devoid of ionizing radiation and can be applied in a broad range of settings .

herein we report the mechanism of oxidative addition of cf3i to au(i ) , and remarkably fast caryl cf3 bond reductive elimination from au(iii ) cations . cf3i undergoes a fast , formal oxidative addition to r3paur ( r = cy , r = 3,5-f2-c6h4 , 4-f - c6h4 , c6h5 , 4-me - c6h4 , 4-meo - c6h4 , me ; r = ph , r = 4-f - c6h4 , 4-me - c6h4 ) . when r = aryl , complexes of the type r3pau(aryl)(cf3)i can be isolated and characterized . mechanistic studies suggest that near - ultraviolet light ( max = 313 nm ) photoinitiates a radical chain reaction by exciting cf3i . complexes supported by pph3 undergo reversible phosphine dissociation at 110 c to generate a three - coordinate intermediate that undergoes slow reductive elimination . these processes are quantitative and heavily favor caryl i reductive elimination over caryl cf3 reductive elimination . silver - mediated halide abstraction from all complexes of the type r3pau(aryl)(cf3)i results in quantitative formation of ar cf3 in less than 1 min at temperatures as low as 10 c .

reports of organogold complexes undergoing redox processes are typically limited to slow oxidative additions and reductive eliminations . however , organogold complexes are not necessarily unreactive ; we recently showed that diaryl au(iii ) complexes undergo remarkably fast aryl aryl reductive elimination at temperatures as low as 50 c . these recent findings from our group , as well those established by vicente , hashmi , and lloyd - jones , suggest that the barrier for challenging reductive eliminations might be substantially diminished at au(iii ) . cf3 bond reductive elimination is typically a slow process requiring elevated temperatures and long reaction times , due to ground state stabilization afforded by exceptionally strong bonding between transition metals and cf3 ligands . for instance , ( dppbz)pd(2-me - c6h4)(cf3 ) ( dppbz = 1,2-bis(diphenylphosphino)benzene ) is stable at 130 c for 3 days , while ( dppp)pd(ph)(cf3 ) ( dppp = 1,3-diphenylphosphinopropane ) and ( dppe)pd(ph)(cf3 ) ( dppe = 1,2-diphenylphosphinoethane ) yield only 10% phcf3 after 3 days at 145 c . reductive eliminations at temperatures between 50 and 80 c can be achieved at pd(ii ) by employing bulky ligands , such as xantphos and brettphos . notably , while aryl - cf3 reductive eliminations from pd(iv ) often require similarly high temperatures , sanford has shown that they can occur at temperatures as low as 23 c over 1 h. despite advances in catalytic trifluoromethylation , caryl cf3 reductive elimination still remains a challenging step . given the importance of trifluoromethylated arenes in pharmaceuticals and agrochemicals , we were prompted to investigate potentially low - barrier caryl cf3 bond reductive elimination at au(iii ) . to access complexes of the type r3pau(aryl)(cf3)i , we were drawn to puddephatt s report of the oxidative addition of cf3i to me3paume to afford cis / trans mixtures of me3paume2(cf3 ) and me3paui . in one case , me3pau(me)(cf3)i was obtained exclusively , but its preparation could not be reproduced by the authors . because reaction times varied from 5 min to 1 day , and rates dramatically slowed in the presence of galvinoxyl , the authors concluded that a free - radical chain mechanism was operative , with cf3 as the propagating species . prior investigations by our group revealed that oxidation of ph3pau(4-f - c6h4 ) rapidly generates 4,4-difluorobiphenyl through a mechanism involving aryl group transfer . however , the use of the bulkier pcy3 prevents transfer of the arene ligand , instead resulting in clean , rapid oxidation of cy3pau(4-f - c6h4 ) ( 1a ) to the isolable au(iii ) complex cis-(cy3p)au(4-f - c6h4)cl2 ( 2 ) ( eq 1).1 therefore , we began our investigations of au(i ) oxidation by cf3i using 1a , with the fluorinated arene ligand also providing a convenient f nmr handle . treatment of 1a in cd2cl2 with cf3i ( 25 equiv ) afforded the product of formal cf3i oxidative addition 3a in 1 h in good yield ( eq 2 and table 1 ) . both the cf3 and pcy3 ligands ( doublet at = 24.5 and quartet at = 25.6 in the f and p nmr spectra , respectively ) provide diagnostic nmr signals ( table 2 ) . the substantial coupling ( jp f = 63 hz ) between fluorine and phosphorus are characteristic of a trans relationship between the cf3 and phosphine ligands . x - ray analysis of crystals of 3a confirmed this stereochemical relationship around the square planar au(iii ) ( figure 1a ) ; other than the homoleptic anion [ au(cf3)4 ] , complex 3a contains a rare example of a crystallographically characterized au(iii)cf3 bond . complex 3a is not only stable to air and water but can be purified by column chromatography as well.2 ( a f ) thermal ellipsoid representations of 3a3d , 12a , and 12b at the 50% probability level . atoms are color - coded : gray ( carbon ) , yellow ( fluorine ) , gold ( gold ) , purple ( iodine ) , orange ( phosphorus ) . the reaction of 1a and cf3i represents a rare oxidation of au(i ) to au(iii ) that directly installs potentially reactive au(iii)carbon bonds . during our attempts to monitor the oxidative addition by f nmr , we found that no reaction occurred when the reaction mixture was placed inside the dark nmr spectrometer . however , when the reaction mixture was exposed to ambient fluorescent light for 5 min , the formation of 3a was detected ( 20% ) . given the reliance of numerous methods on cf3i as a trifluoromethyl source , we investigated its photochemical reactivity . actinometry experiments were carried out to determine the overall quantum yield , using the norrish ii fragmentation of valerophenone as a standard . the oxidative addition of cf3i to 1a was complete after 20 s of irradiation by a hg vapor lamp ( 2 mm aq . k2cro4 optical filter ; transmittance max = 313 nm ) , while the fragmentation of valerophenone ( = 1 ) took place over 24 h under identical conditions . this rate difference , in addition to the ability of ambient light to bring the reaction to full conversion over variable reaction times ( between 15 min and 1 h ) , supports a radical chain reaction as the mechanism of au(i ) oxidation by cf3i . the reaction of excess cf3i and 1a is also fast in thf , but the conversion is never greater than 65% ( 52% yield of 3a ) , even when irradiated by a hg vapor lamp for 1 h ( vida infra ) . notably , an excess of fluoroform ( hcf3 ) is generated in thf , regardless of the light source ( only dcf3 is formed when thf - d8 is used ) . gc - ms analysis of reaction mixtures reveals several products of thf oxidation , likely formed by h abstraction by cf3 . several control experiments , using hcf3 production relative to a standard as a probe to detect cf3 generation , support the involvement of au(i ) during the initiation of the chain reaction . the uv absorption of cf3i is centered at 270 nm but tails beyond 350 nm . when irradiated at 313 nm , cf3i undergoes fast , reversible c however , in the absence of 1a , only negligible amounts of hcf3 are observed when thf solutions of cf3i are irradiated for 30 min , indicating that carbon / iodine radical recombination is substantially faster than h abstraction from thf . similarly insignificant quantities of hcf3 are observed when 20 equiv ( relative to cf3i ) of the h donors 1,4-cyclohexadiene , 9,10-dihydroanthracene , or triphenylmethane are added ( figure 2a ) . additionally , cy3pau(2-(ch2ch = ch2)c6h4 ) ( 4 ) , containing a pendent olefin to either capture a putative au(ii ) intermediate and/or cf3 , is fully consumed upon irradiation in the presence of excess cf3i ( figure 2b ) . this oxidation affords multiple au(iii ) products of indiscriminate cf3 addition to the terminal olefin and gold atom ( and hcf3 when thf is used as solvent ) ( see si ) . because 2-allylbromobenzene ( 5 ) does not react with cf3i when irradiated under similar conditions ( no hcf3 is observed after 5 min , and less than 2% after 30 min ) , we conclude that the au(i ) aryl complex is necessary for chain initiation . these results are also consistent with an initiation mechanism involving [ cf3i ] , which generates iodide and cf3 following c i bond homolysis . control experiments to assess involvement of au(i ) in the initiation of the radical chain mechanism . ( a ) irradiation of cf3i solutions containing h donors to detect cf3h in the absence of gold . ( b ) radical trapping using an olefin with and without a pendant gold center . we envisioned two possible initiation mechanisms for generating cf3 as a propagating species from [ cf3i ] in a chain reaction : ( 1 ) initial photoexcitation of 6 followed by electron transfer to cf3i , or ( 2 ) initial photoexcitation of cf3i followed by electron transfer from 6 ( scheme 1 ) . au(i ) aryl complexes are well - known chromophores , and their photophysical properties have been investigated previously . while 1a absorbs weakly above 310 nm ( the cutoff for many laboratory fluorescent lamps ) , excitation at 320 nm ( = 37 m cm ) results in a weak , broad luminescence from 340 to 460 nm , classified as fluorescence based on the lifetime of excited species 1a * ( < 10 ns , quantum yield of fluorescence = 0.03 ) . despite the short lifetime of 1a * , cf3i effectively quenches its fluorescence ( stern volmer quenching constant ksv = 30 m , figure 3 ) . although this energy transfer could conceivably generate cf3 and initiate a chain reaction ( mechanism 1 , scheme 1 ) , when cf3i is removed from fluorimetry samples under vacuum , fluorescence is restored to the same intensity prior to introduction of the gas , indicating that consumption of au(i ) has not occurred . volmer plots of fluorescence quenching of 1a by different concentrations of cf3i ( blue boxes ) and au(iii ) complex 3a ( blue triangles ) in ch2cl2 . concentrations of au(iii ) are in mol / l and cf3i concentrations are in mmol / l . surprisingly , fluorescence quenching by the au(iii ) complex 3a is more than 2 orders of magnitude more effective ( ksv = 4270 m ) than quenching by cf3i ( figure 3 ) . if propagating species terminate frequently , some critical concentration of au(iii ) product exists that may impede productive energy transfer from an excited species , halting reinitiation of the chain reaction . in light of puddephatt s report , au(i ) alkyl complexes , such as me3paume , clearly react with cf3i . however , there is no mention of the dependence of light on this process , although if the reaction is photoinitiated , mechanism 1 would seem especially unlikely given the absence of a chromophoric aryl ligand in puddephatt s examples . to test this hypothesis , we irradiated cy3paume ( 9 ) in the presence of cf3i ( scheme 2 ) . while 9 does not absorb above 300 nm ( see si ) , the reaction is quantitative in cd2cl2 when irradiated with ambient light , and does not proceed in the dark . the oxidized product is unobservable , eliminating ch3i to generate cy3paucf3 at room temperature . in thf , the reaction generates excess hcf in thf3 , presumably also from solvent h abstraction by cf3 . if initiation mechanism 2 is operative , then cf3 could be generated by irradiating cf3i solutions containing electron donors other than au(i ) , such as phosphines ( scheme 3 ) . indeed , irradiation of pme3 or pcy3 in the presence of cf3i results in formation of [ me3p - cf3]i ( 10a , jp consistent with quenching of [ cf3i ] * by au(iii ) , the oxidation of pcy3 in thf stalls at roughly 45% conversion ( by p nmr ) in the presence of 25 mol % au(iii ) complex 3a . pph3 does not react with cf3i ( eq 3 ) , presumably due to its lower oxidation potential relative to pme3 and pcy3 . when pcy3 and pph3 are irradiated together with cf3i , only pcy3 is consumed , suggesting that pph3 neither initiates the chain nor reacts with cf3 during propagation . contrary to our initial hypothesis that bulky phosphine ligands prevent aryl group transfer upon au(i ) oxidation , we found that ph3pau(4-f - c6h4 ) ( 11a ) undergoes quantitative photoinitiated reaction with cf3i in cd2cl2 to generate 12a ( eq 4 ) . since pph3 is unreactive toward cf3i , oxidation of 11a can not be initiated by small amounts of dissociated pph3 ( we can not disprove the analogous mechanism for pcy3-supported complex 1a . ) complex 12a was characterized by x - ray crystallography and shown to be isostructural to 3a ( figure 1b).3 on the basis of these results , we propose that while photoexcited [ cf3i ] * undergoes rapid c i bond homolysis and recombination , it also oxidizes au(i ) aryl and alkyl complexes by accepting electrons into a low - lying somo to generate radical anion [ cf3i ] ( mechanism 2 , scheme 1 ) . homolysis of the c i bond of [ cf3i ] generates iodide and cf3 , which oxidizes ( r3p)aur ( 6 ) to au(ii ) intermediate 7 . iodine atom abstraction of cf3i by 7 affords au(iii ) complex 8 and regenerates cf3 . in thf , oxidation of 6 by cf3 is competitive with solvent h abstraction to make hcf3 and terminate the radical chain . at sufficiently high concentrations , the au(iii ) product ( 8) quenches [ cf3i ] * before it can reinitiate the radical chain reaction promisingly , the photoinitiated oxidative addition of cf3i is general for electronically diverse complexes of the type cy3pau(aryl ) ( tables 1 and 2 ) . the resulting au(iii ) products ( see figure 1 for their crystallographic analyses ) can be purified by chromatography on silica . complex 1b ( aryl = 3,5-f2-c6h3 ) , which is more electron - deficient than 1a ( aryl = 4-f - c6h4 ) , reacts smoothly with cf3i to afford 3b . while complexes with more electron - rich ligands such as 1c ( aryl = c6h5 ) and 1d ( aryl = 4-me - c6h4 ) also react with cf3i to afford 3c and 3d , respectively , the most electron - rich complex 1e ( aryl = 4-meo - c6h4 ) decomposes to au nano particles and several cf3-containing au(iii ) complexes in solution and solid state ( no products of caryl au(iii ) product 3e is detectable , however , and its decomposition can be slowed substantially by addition of mecn upon concentration of the reaction , allowing its solution - state characterization . the mechanism of decomposition has not yet been identified , although we speculate that the electron - rich arene may encourage pcy3 dissociation at room temperature and subsequent aryl group transfer . the complex 1f ( aryl = 2-me - c6h4 ) does not react with cf3i at all , suggesting that cf3i oxidative addition is sensitive to the sterics of the aryl ligand and that relaxation of [ cf3i ] * is faster than oxidation of the metal center to initiate the radical chain . unsurprisingly , no hcf3 is observed when 1f is irradiated in thf for 20 min . cf3 reductive eliminations from au(iii ) . to our surprise , 12a undergoes quantitative caryl i reductive elimination in toluene - d8 at 110 c to afford 4-fluoroiodobenzene and ph3paucf3 over 20 min ( scheme 4 ) . this process is highly sensitive to free phosphine , stalling completely in the presence of pph3 ( 0.1 or 1.0 equiv ) at 110 c for 12 h. treatment of 12a with pph3-d15 at room temperature results in immediate formation of 12a - d15 , presumably via an associative process . more electron - rich aryl ligands , such as 4-methylphenyl ( 12b ) , do not significantly affect the relative rates of caryl i and caryl cf3 reductive elimination ( scheme 4 ) . at 110 c , complex 12b undergoes mostly caryl i reductive elimination within 10 min to afford 4-methyliodobenzene . both caryl i and caryl cf3 reductive eliminations are also completely inhibited in the presence of pph3 ( 0.1 or 1.0 equiv ) , while pph3-d15 reacts immediately at room temperature to afford 12b - d15 , also via associative ligand exchange . these observations are consistent with a mechanism involving highly reversible pph3 dissociation from 12a and 12b , followed by slow caryl , the behaviors of 12a and 12b are similar to au(iii)alkyl complexes studied by kochi , which not only reductively eliminate calkyl calkyl bonds between 70 and 100 c via a dissociative mechanism but also undergo associative ligand exchange at ambient temperature with excess phosphine . unsurprisingly , analogous pcy3-stabilized complexes 3a and 3d are stable at 110 c for at least 12 h , presumably due to the greater -donating ability of pcy3 relative to pph3 . phosphine exchange with excess p(n - bu)3 , pbn3 , or pcy3 does not occur even at these temperatures , precluding not only the lower - barrier associative exchange mechanism observed with the pph3-supported systems ( attributed to the larger cone angle of pcy3 relative to pph3 ) , but also pcy3 dissociation to form a three - coordinate complex . because caryl i reductive elimination is significantly faster than caryl cf3 reductive elimination , a cycle for gold - catalyzed trifluoromethylation must necessarily involve iodide abstraction from the au(iii ) product of cf3i oxidative addition . despite the apparent kinetic stabilities of the au(iii ) complexes 3a3e , 12a , and 12b , they all undergo quantitative caryl cf3 reductive elimination in less than 1 min upon treatment with agsbf6at room temperature . to consider the effects of the phosphine ligand on the silver - mediated caryl cf3 reductive elimination of au(iii ) , we used variable - temperature nmr to follow the reductive elimination from 3a and 12a in the presence of agsbf6 . pcy3-substituted complex 3a undergoes very fast ( quantitative conversion in less than 1 min ) caryl cf3 reductive elimination at 10 c , while the analogous pph3-stabilized 12a reacts similarly fast at room temperature ( eq 5 ) . at lower temperatures , several bridging species ( most likely dimers ) are observed by f nmr upon halide abstraction in both cases . if caryl cf3 bond reductive elimination can only occur from a monomeric three - coordinate intermediate , then 12a might be expected to undergo slower reductive elimination due to slower dimer dissociation and/or a dimer monomer equilibrium that more favors the dimer , based on the smaller cone angle and weaker -donation of pph3 relative to pcy3.5 these results reported herein support the oxidative addition of cf3i to au(i ) via a photoinitiated chain reaction . the reactions are fast at room temperature for both au(i ) aryl and alkyl complexes . aryl - cf3 reductive elimination is typically a high - barrier process but occurs in seconds at room temperature from a au(iii ) cation . the au(i)aryl species may be regenerated via one of the numerous transmetalation strategies available involving carbon nucleophiles . for instance , excess ( 4-f - c6h4)snme3 ( 10 equiv ) undergoes fast , quantitative transmetalation with [ cy3pau]sbf6 at room temperature to afford 1a , thereby closing a hypothetical catalytic cycle based on the three elementary steps shown in scheme 5 . silver - free halide abstraction from au(iii ) complexes could conceivably enable a practical and mild cycle for gold - catalyzed trifluoromethylation of aryl nucleophiles , although deleterious reactions between starting material and metalloradical intermediates and cf3 must be mitigated , as well as competitive aryl aryl homocoupling . while we initially set out to probe caryl cf3 reductive elimination at au(iii ) , we also explored the oxidative addition of cf3i to au(i ) , a process with potential implications beyond gold chemistry . the possibility of photoinitiated oxidation of transition metals or main group elements by cf3i should not be discounted in methods employing this reagent as a trifluoromethyl source , particularly since ambient fluorescent laboratory lighting is sufficient to initiate a chain in the presence of a suitable reductant . the results presented also suggest that substrate photoexcitation may provide a low - barrier avenue to kinetically challenging oxidative additions by au(i ) , providing access to potentially reactive au(iii ) complexes .

for tissue diagnosis of suspected pancreatic cancer , endoscopic ultrasound - guided fine needle aspiration ( eus - fna ) is the procedure of choice with high safety and accuracy profiles . however , about 10% of cytologic findings of eus - fna are inconclusive . in that situation , careful observation , surgical exploration , or alternative diagnostic tools such as bile duct brushing with endoscopic retrograde cholangiopancreatography or computed tomography - guided biopsy can be considered . however , some concerns and/or risks of these options render repeat eus - fna a reasonable choice . repeated eus - fna may impose substantial clinical impact with low risk .

despite technical advancement of various imaging modalities , it is still impossible to differentiate benign and malignant pancreatic lesions by the images only . for tissue acquisition to differentiate pancreatic lesions , endoscopic ultrasound - guided fine needle aspiration ( eus - fna ) is the procedure of choice with high accuracy and low complication rate . pooled sensitivity and specificity of eus - fna in the diagnosis of etiology of solid pancreatic mass was 86.8% and 95.8% , respectively , in a meta - analysis.1 although eus - fna shows high diagnostic accuracy in patients with suspected pancreatic carcinoma , the rate of acquiring indeterminate cytologic findings is still up to 10.9%.2 endosonographer face difficulties when cytology result of eus - fna is inconclusive while malignancy is highly suspicious in clinical presentation . although eus is minimally invasive , very safe and accurate technique for tissue diagnosis , sometimes endosonographers get nondiagnostic eus - fna results . after obtaining specimen by eus - fna , cytopathologist reads the sample and classifies them into inadequate , benign / reactive , atypical , suspicious , and/or malignant . problems of eus - fna are inconclusive results and diagnostic errors including false positive and false negative . the reasons for those problems stem from the various situations such as failed puncture , successful puncture but obtained inadequate sample material , or successful puncture , obtained adequate sample material but cytology was negative for malignancy . there are several options . in the first place , clinical observation and follow - up with serial imaging one study showed that about 30% of patients with negative or nondiagnostic eus - fna result were finally able to be diagnosed as pancreatic cancer later.3 in that situation , visible predictors of malignancy on the eus were vascular invasion or lymphadenopathy.3 when pancreatic cancer is clinically suspected , careful short - term follow - up with eus or other imaging modalities is very risky . next , surgical exploration with blind pancreatic resection or chemoradiation therapy without definitive tissue diagnosis may be an option . when endosonographers get negative or nondiagnostic eus - fna result while pancreatic cancer is highly suspicious clinically , the most beneficial next step for the patient should be sought . alternative diagnostic tools would be chosen to get tissue for diagnosis , such as bile duct brushing with endoscopic retrograde cholangiopancreatography ( ercp ) or computed tomography ( ct)-guided biopsy . however , ercp with brushing is associated with postprocedural complications such as pancreatitis and ct - guided biopsy bears the risk of intraperitoneal spread . one study compared alternative methods which can be utilized for tissue sampling when pancreatic cancer is suspicious.4 ercp with brushing showed low sensitivity and surgical biopsy showed very high complication rate . ct or abdominal ultrasound - guided percutaneous approach showed high rate of failure compared with eus - fna . considering that all of these options have some concerns and/or risks , retrial of eus - fna can also be a reasonable option . if we can change one of the components of eus - fna , we can anticipate different results of repeated eus - fna . factors that can influence the result of eus - fna related with lesion are location , characteristics and size . as to location , pancreatic head and uncinate are even more difficult area.5 though we can not change the location of the lesion , sometimes we can approach pancreatic head through the stomach , rather than the duodenum to get a better result . characteristics of the lesion are also an important factor . if there is background pancreatitis , or extensive necrosis of the mass , it is difficult to get a good result from eus - fna.6 we may try to target different area of the lesion to get a better result . accuracy of eus - fna increases as the size of the pancreatic mass increases.7 however , one interesting study suggests that repeated eus - fna can improve diagnostic yield even in small pancreatic masses.8 they evaluated the role of repeated eus - fna for small pancreatic mass with previous indeterminate and negative cytology . from january 2004 to october 2006 , 47 eus - fna was done for pancreatic mass of less than 3 cm in size . initial eus - fna results were 17 malignancies , 21 benign , and nine indeterminate . repeated eus - fna for these nine indeterminate cases resulted in six malignancies , one benign , and two indeterminate . again , eus - fna was done for these two indeterminate cases and one malignancy was proved . as a consequence , initial diagnostic accuracy of eus - fna 83% was increased up to 96% by repeated eus - fna . regarding equipment , ultrasound processor with better resolution can give us better view of targeting the lesion , but there is no study comparing the efficiency of ultrasound processor for eus - fna . there is suggestion that newly developed forward viewing echoendoscope may be helpful to puncture difficult lesions such as uncinate process or head of the pancreas.9 there will be articles about eus - fna needles in this issue of clinical endoscopy . eus - fna is a technically demanding procedure with a steep learning curve.10 this means that the result of eus - fna may be very operator - dependent . according to guidelines for credentialing and granting privileges for eus by american society for gastrointestinal endoscopy , minimum of 150 supervised cases is recommended for competency of eus . to perform eus - fna , at least 25 supervised eus - fna is recommended.11 but a study showed that the rate of positive yield of eus - fna is increasing after 20 , 30 , 40 eus - fna procedures.12 after looking at the learning curve of 300 consecutive eus - fna procedures , a study suggested that even after 45 eus - fnas during fellowship , more procedures are needed to gain proficiency and efficiency with eus - fna . these studies teach us that the yield of eus - fna depends on the experience of endosonographer.13 a well - trained cytopathologist is an essential element of successful eus - fna procedure and presence of on - site pathologist in the endoscopy suite and rapid on - site cytopathological examination are very helpful to get adequate specimen . on the other hand , an interesting study revealed that cytopathologists ' expertise could impact the diagnostic accuracy of eus - fna result . in that study , local cytopathologists mailed the eus - fna slides of difficult cases to expert cytopathologists . diagnostic sensitivity and accuracy were 72% and 75% for local cytopathologists , respectively , and 89% and 88% for expert cystopathologists , respectively.14 when eus was repeated for a similar clinical indication at a tertiary - referral center , a significant clinical impact was observed in 63% of the patients.15 repeated eus at the same center with various indications , also resulted in change of further management plan in 72% of the patients.16 from those results , we might expect that repeated eus - fna by expert at another center or by the same endosonographer with different setting can give successful result as the cases of colonoscopy or ercp maybe successfully performed on the previously failed procedure . a study reported overall accuracy of second eus - fna as 84%.6 twenty four repeat eus - fna were done in the study center and second eus - fna proved malignancy in 46% of the cases . eight out of 10 atypical / suspicious cases with initial eus - fna confirmed malignancy . surprisingly , two out of 10 benign initial eus - fna cases changed diagnosis to malignancy and two malignancy cases were confirmed as benign . another researcher performed repeat eus - fna 3 weeks later , when initial eus - fna result was indeterminate for solid pancreatic lesion . the result proved that 78% ( 7/9 ) of the patients with indeterminate cytology results had malignancy.8 when eus - fna was performed in 62 cases of repeated eus , 82% of patients ( 47/62 ) among them had inconclusive cytology with previous eus - fna , 73% cases ( 45/62 ) were prevented from undergoing further diagnostic work - up.16 there is a retrospective study that evaluated repeated eus - fna performed in 15 cases including eight pancreatic mass and seven unknown intra - abdominal lymphadenopathy . second eus - fna proved malignancy in 60% of the cases and overall accuracy of second eus - fna was 92.9%.17 mean while , there is a study of low diagnostic yield of repeat eus - fna . twenty eight repeat eus - fna were done when pancreatic cancer was suspected but prior eus - fna results were non - diagnostic . repeating eus - fna is a reasonable choice . repeated eus - fna may impose substantial clinical impact with low risk . in clinical practice , repeated eus - fna is useful when the initial eus - fna result of a suspected tumor is nondiagnostic . repeat eus - fna should be considered especially if predictors of malignancy , such as vascular invasion or lymphadenopathy , are visible on the eus .

this study examined the changes in cellular glucose uptake induced by 2,3,7,8 tetrachlorodibenzo - p - dioxin ( tcdd ) as measured by quantification of intracellular radioactivity in the nih 3t3 l1 preadipocyte cell line after a 30-minute incubation with the non - metabolizable radioactive analogue of glucose , 3-o - methyl - d-[1 - 3h ] glucose . treatment of differentiated nih 3t3 l1 cells with tcdd produced a time- and dose - dependent decrease in the cellular uptake of glucose . treatment of cells for 3 hr with 10(-8 ) m tcdd significantly reduced glucose uptake to about 10% of control values ( p < /= 0.05 ) . furthermore , cytochalasin b , a specific inhibitor of facilitative glucose transporter proteins totally abolished the portion of glucose transport activity that is sensitive to tcdd . the role of the ah receptor in tcdd - mediated reduction in glucose uptake was investigated . pretreatment of 3t3 l1 cells with the ah receptor blocker 4,7-phenanthroline antagonized the effect of tcdd on glucose uptake . structure - activity relationship studies with tcdd and two polychlorinated biphenyl ( pcb ) congeners revealed a rank order for their potency in the inhibition of glucose transport as follows : tcdd < < 3,3',4,4 ' tetrachlorobiphenyl < 2,2',5,5 ' tetrachlorobiphenyl ( tcb ) . such a rank order correlates both with previously determined biological activity of tcdd and the more active 3,3',4,4'- and less active 2,2',5,5'-tcb and with affinity for binding to the ah receptor . the thyroid hormone t4 , like tcdd , reduced glucose uptake and blocked the action of tcdd to further reduce glucose uptake . experimental evidence is consistent with a proposed mechanism for tcdd to reduce the titer of functional glucose transporter proteins through its interaction with the ah receptor.imagesp454-afigure 1.figure 2 .

three assays were developed to enumerate total and fecal escherichia coli and total vibrionaceae in shellfish , seawater , and other foods and environmental samples . assays involve membrane overlays of overnight colonies on nonselective agar plates to detect -glucuronidase and lysyl aminopeptidase activities for e. coli and vibrionaceae , respectively . cellulose membranes containing the substrates 4-methylumbeferyl--d - glucuronide ( mug ) produced a bright blue fluorescence when overlaid onto e. coli , while l - lysyl-7-amino-4-trifluoromethylcoumarin produced green fluorescent foci when overlaid onto vibrionaceae family members . a multiplex assay was also developed for simultaneously enumerating total e. coli and total vibrionaceae in oysters and seawater . overall , 65% of overlaid e. coli ( non - o157:h7 ) were mug - positive , compared with 62% as determined by the most - probable - number - mug assay . the overlays are rapid , simple , and cost effective for quantification purposes . this research provides practical alternatives for monitoring bacterial indicators and potential pathogens in complex samples , including molluscan shellfish .

a technique known as the colony overlay procedure for peptidases ( copp assay ) was previously developed as the first test to enumerate total vibrionaceae in shellfish , seawater , and well water [ 1 , 2 ] . this assay has been recommended for use in monitoring molluscan shellfish and for potentially regulating their harvest based on the levels of total vibrionaceae . this total vibrionaceae approach is similar in concept to traditional sanitary surveys of shellfish harvesting areas , which are based on the total number of fecal coliforms or e. coli present in either the shellfish or their surrounding waters [ 3 , 4 ] . in the original copp assay , a substrate , l - lysyl-7-amino-4-trifluoromethylcoumarin ( l - lys - afc ) , is bound to a cellulose acetate membrane and the membrane is overlaid for 10 minutes onto an overnight culture grown on nonselective agar media . a lysyl aminopeptidase that is present in all vibrionaceae family members tested to date cleaves the substrate to produce fluorescent foci on the membrane when viewed under longwave uv . in the present study , we extended this overlay concept to produce similar assays for total and fecal e. coli . these assays are based on the presence of -glucuronidase ( gud ) activity , which is found in most non - o157:h7 e. coli and results in cleavage of the substrate 4-methyl--d - glucuronide ( mug ) into the fluorescent 4-methylumbelliferone . mug - based fluorescence assays for e. coli have been previously developed for food and water [ 612 ] . mug - based assays of foods and particularly molluscan shellfish often involve the most probable number ( mpn ) procedure , which is labor intensive , relatively costly , and requires up to three days for results to be obtained . consequently , simpler , more rapid , less expensive , and more direct enumerative procedures are needed to monitor for total and fecal e. coli , particularly in perishable foods , like shellfish . in this study , we report on simple membrane overlay methods to separately enumerate total e. coli ( capable of growing at 37c ) , fecal e. coli ( capable of growing at 44.5c ) , and total vibrionaceae ( capable of growing at 37c ) , and a fourth multiplex method to enumerate total e. coli and total vibrionaceae simultaneously . these methods are applicable for use in monitoring the presence of these organisms in shellfish and other foods , water , and environmental samples . stock cultures consisted of 37 strains of non - o157:h7 e. coli , as described in table 1 , and 22 enterobacteriaceae within the genera citrobacter , klebsiella , pseudomonas , salmonella , shigella , serratia , and yersinia ( table 2 ) . human pathogenic vibrios consisted of v. cholerae o1 and v. vulnificus ( vv1003 ) , as previously described , and v. parahaemolyticus o3:k6 , a pandemic strain obtained from e. fidelma boyd at the university of delaware , newark , delaware . eastern oysters ( crassostrea virginica ) were obtained from a commercial source in rhode island and from a tidal river at the university of delaware marine laboratory in lewes , delaware . the oysters were naturally contaminated with low levels of e. coli and moderate levels of vibrionaceae . they were collected principally during the warm , summer months when vibrionaceae were present in the water column . seawater was also obtained from the university of delaware marine laboratory during the summer . the mpn procedure using mug in the ec broth tubes was performed on dilutions of pure cultures and on seawater and oyster homogenates according to the protocol published in the u.s . the mug membrane overlay procedure was developed and its specificity was determined using e. coli serotypes and other enterobacteriaceae as listed in tables 1 and 2 . the bacteria were grown in tryptic soy broth ( becton , dickinson and co. , sparks , maryland ) supplemented with an additional 0.5% nacl ( 1% total nacl ) and were streaked onto 100 mm plates of tryptic soy agar ( becton , dickinson and co. ) containing a total of 1% nacl ( tsa - n ) using a flamed and cooled metal triangle . plates were incubated overnight ( ~18 hours ) at 37c . after culture incubation , cellulose chromatography papers ( whatman international , maidstone , uk ) or whatman no . 1 , 4 , or 54 cellulose filter paper discs ( referred to as cellulose membranes ) were cut to the desired size ( of the petri dish or colonies to be overlaid ) and soaked in 20 mm tris buffer , ph 8.0 containing 50 mg / l mug for about 10 seconds . excess buffer was allowed to drip from the membranes for 5 seconds , and the membranes were overlaid onto colonies growing on the surface of the tsa - n plates . unused substrate was retained at 4c for future use and has a shelf life of several weeks . membranes were removed with forceps and placed in empty petri dishes and examined for fluorescent foci using either a hand - held uv lamp at a wavelength of 364 nm or a uv light box at the same wavelength . alternatively , membranes could be viewed with the hand - held uv lamp while still overlaying the colonies . the copp assay was performed with cellulose acetate membranes as previously described [ 1 , 2 ] or with cellulose chromatography paper ( whatman international ) . in essence , a substrate l - lysyl-7-amino-4-trifluoromethylcoumarin ( l - lys - afc ; cat . afc-008 , mp biomedicals , salon , oh ) was dissolved in dimethylsulfoxide ( dmso ) to 20 mm ( 2140 l of dmso to 25 mg of l - lys - afc ) and the stock was stored at 20c until needed . a 250 m substrate working solution was prepared by combining 25 l of thawed substrate and 2 ml of 20 mm tris , ph 9.0 . substrate stock solution may be refrozen and thawed multiple times without appreciable deterioration of the substrate . a cellulose acetate membrane or a plain cellulose membrane ( chromatography paper ) was cut to the desired size and placed in the working solution for 1015 seconds . the membrane was lifted with forceps , allowed to drip excess fluid for 5 seconds , and overlaid onto overnight bacterial colonies grown at 37c on tsa - n plates . each membrane was removed from the plate , placed in a clean petri dish , immediately viewed under longwave ( 364 nm ) uv , and fluorescent foci were counted . alternatively , the foci could be visualized with a hand held uv lamp while the membrane was still covering the colonies . the appearance of bright white or pale blue fluorescence on cellulose acetate membranes or green fluorescence on cellulose chromatography paper signifies the presence of a lysyl aminopeptidase , which cleaves the substrate . positive controls consisted of v. cholerae , v. parahaemolyticus , and v. vulnificus and were propagated at 37c . the overlay of enterobacteriaceae was also performed at 37c for 10 minutes to determine if any of the species contained a lysyl aminopeptidase activity similar to that found in the vibrionaceae . a method to simultaneously detect total vibrionaceae and total e. coli was evaluated using cellulose chromatography paper that was soaked in 20 mm tris , ph 8.0 containing 250 m l - lys - afc and 50 mg / l mug . oysters were either 1 : 10 homogenates or dilutions of the homogenates that were prepared in 0.1% peptone buffer . plates were prepared using 100 l of each homogenate or dilutions of the homogenates , which were spread over tsa - n plates with a flamed and cooled metal triangle . after overnight incubation at 37c , membranes containing both substrates were overlaid onto the culture plate . vibrionaceae were enumerated in situ after 10 minutes followed by the enumeration of mug - positive ( gud - positive ) colonies after an additional 20 minutes ( 30 minutes total ) . it was anticipated that total e. coli could be easily distinguished from total vibrionaceae based on the color of the fluorescent foci . forty - three principally food - borne outbreak strains of e. coli o157:h7 were screened for gud activity by both the mpn - mug assay and by the mug overlay of colonies that were grown on tsa - n and incubated at 37c for 2 hours followed by 44.5c overnight . plates were overlaid for 30 minutes and the membranes were viewed on a uv light box . oysters and seawater were screened by the membrane overlay assay for total e. coli and total vibrionaceae and by multiplex assays for both by spread plating 100 l of a 1 : 10 or 1 : 100 oyster homogenate , prepared in 0.1% peptone buffer , or 100 l of undiluted seawater onto tsa - n plates . the plates were allowed to incubate at either 37c for total e. coli and total vibrionaceae overlays or at 37c for 2 hours followed by 44.5c overnight for e. coli overlays using cellulose membranes containing mug and copp separately and combined . samples were occasionally plated in duplicate and one plate incubated at 37c for the multiplex assay of total vibrionaceae and total e. coli while the other plate was incubated at 37c for 2 hours and then at 44.5c overnight for fecal e. coli enumeration . initial tests showed that overnight colonies of e. coli growing on tsa - n plates could be overlaid with cellulose membranes ( chromatography paper or whatman no . 1 , 4 , or 54 filter paper discs ) soaked in mug to produce strong blue fluorescent foci on the membranes , corresponding with the site of contact between the colony and the membrane . comparable results were obtained regardless of which type of membrane was used . consequently , we selected the chromatography paper to perform mug overlays throughout this study . an evaluation of substrate concentrations showed that strong fluorescence intensity was achieved when membranes were saturated in 50 mg / l mug . membranes were incubated for 1560 minutes , and then viewed for fluorescent foci in situ with a hand - held uv lamp , or the membrane could be removed to a petri dish and viewed with the hand - held uv lamp or placed on a uv light box for viewing . optimal results were obtained after a 3060-minute overlay and 30 minutes was selected for the remainder of the study . in contrast , colonies to be screened for total vibrionaceae by the copp assay were saturated in 250 m of l - lys - afc and overlaid for only 10 minutes to preclude weak enzyme activity , which is present in some enterobacteriaceae and other non - vibrionaceae , from producing false positive fluorescent foci . figures 1(a)1(d ) show mug and copp fluorescence on cellulose membranes after overlaying colonies of three serotypes of non - o157 : h7 e. coli ( top row in each panel ) and three vibrionaceae ( bottom row in each panel ) . edges of the foci are not distinct and sharp , but somewhat fuzzy because enzyme activity is via diffusion of the enzymes within the membranes . examples of representative mug fluorescence on the membranes are shown for total e. coli that had been incubated at 37c overnight and overlaid for 30 minutes with mug - containing membranes ( figure 1(a ) ) , and for fecal e. coli grown at 37c for 2 hours , incubated overnight at 44.5c , and overlaid for 30 minutes with a mug membrane ( figure 1(b ) ) . these same vibrios are strongly copp positive after overnight incubation at 37c and overlay for 10 minutes with l - lys - afc , producing green fluorescence on cellulose membranes , as depicted in figure 1(c ) . very weak copp fluorescence is occasionally observed in the e. coli ( arrow , figure 1(c ) ) , and such foci should not be counted as vibrionaceae because of the weak signal . this fluorescence may be minimized by enumerating the foci immediately after the 10-minute overlay . results of the multiplex assay on cultures incubated at 37c overnight , followed by a 30-minute overlay with mug and l - lys - afc ( combined ) are shown in figure 1(d ) with blue fluorescence from the e. coli on the top row and green fluorescence from the vibrios on the bottom row . the arrow in figure 1(d ) indicates the blockage of uv light from an opaque colony of v. parahaemolyticus which inadvertently transferred to the membrane and blocked the passage of the uv light from the light box through the membrane . such transfer of opaque colonies to the membranes occasionally occurs and appears to be a function of the stickiness of the colony . in such cases , foci may be readily observed by holding a hand - held uv lamp on the opposite side of the membrane ( the side without the opaque material ) . the mug overlay and the mpn - mug assays were compared for 37 strains of e. coli ( non - o157:h7 ) . twenty - four ( 65% ) of the 37 strains were positive by the mug overlay and 23 ( 62% ) were positive by the mpn - mug assay ; however , four colonies that were mug - negative by overlay were positive or weakly positive by the mpn - mug method , specifically strains o14:nm , o42:h2 , o103:h2 , and o111:nm ( table 1 ) . likewise , five isolates that were mug - negative according to the mpn - mug method were positive by the overlay method , specifically one each of the o4:nm and o26:h11 strains , and o78:h11 , o91:h , and o91:h14 ( table 1 ) . mpn - mug often gave weak fluorescence signal ( listed as in table 1 ) . repeat assays of these isolates showed variability in the perceived fluorescence intensities for those cultured in mpn - mug media . no variability was seen on the overlays , which consistently produced bright blue foci . copp overlays of these cultures were performed for 10 minutes with readings taken immediately after overlay , and weakly positive results were obtained for four of the isolates ( table 1 ) . differentiation of the shigella spp . was possible using individual mug and copp assays where one sh . boydii was both mug and copp positive , sh . flexneri was both mug and copp negative , sh . dysenteriae was mug negative and copp positive , and sh . sonnei was mug positive and copp negative with cultures propagated at 37c ( table 2 ) . shigella boydii , sh . dysenteriae , and y. enterocolitica were copp positive indicating an enzyme analogous to the lysyl aminopeptidase that was found in vibrionaceae family members . this is the first time we detected strong copp positive colonies associated with bacteria that were unrelated to the vibrionaceae . forty - three e. coli o157:h7 strains were tested in triplicate and were mug negative by both the overlay and mpn - mug assays ( data not shown ) . it is well known that o157:h7 strains lack gud activity and are therefore mug negative with few exceptions . membranes simultaneously soaked in mug and l - lys - afc were used in a multiplex format to detect total e. coli and total vibrionaceae on plates that were grown overnight at 37c ( figures 1(e ) and 1(f ) ) . in the multiplex assay of oyster homogenates , green and dark blue foci are obtained for vibrionaceae and total e. coli , respectively , when incubated at 37c ( figures 1(e ) and 1(f ) ) . for accurate enumeration , fluorescent foci must be counted in stages . membranes were incubated for 10 minutes on the plates and viewed with a hand - held uv . green fluorescent foci , representing the vibrionaceae colonies , were counted immediately , to avoid low levels of enzymes in some non - vibrionaceae from leading to false positive results . the plate was then incubated for an additional 20 minutes or longer at 37c for blue fluorescence development , which is indicative of colonies of total e. coli . for illustration purposes only , membranes shown in figures 1(e ) and 1(f ) were illuminated on a uv light box and photographed after a 30-minute overlay , which was sufficient to give bright blue fluorescence from the gud - positive colonies but was longer than recommended for accurate vibrionaceae enumeration . simultaneous detection is only practical if countable levels of total e. coli and total vibrionaceae are present on the same dilution plate , although figures 1(e ) and 1(f ) demonstrate the ease with which total e. coli colonies may be identified , even on crowded plates . multiplex assays are not appropriate for cultures incubated at 44.5c , since many vibrionaceae may not grow at elevated temperatures . one exception was our v. cholerae o1 which produced typical copp fluorescence when grown at 44.5c . if total vibrionaceae and total and fecal e. coli enumeration are desired for a sample , duplicate plating allows one plate to be incubated at 37c for total vibrionaceae and total e. coli while the other plate is incubated at 37c for 2 hours and then at 44.5c overnight for fecal e. coli enumeration . the 2 hours incubation at 37c constitutes a resuscitation step , as oysters may contain stressed bacteria which need to be resuscitated . higher e. coli counts were obtained in our assays when the resuscitation step was employed . from a safety standpoint , the mug assay offers additional protection against pathogens , by detecting some strains of salmonella and shigella which were mug positive and could grow at 44.5c , specifically , s. montevideo , sh . seawater produced bacterial colonies that gave a fluorescence signal comparable to that found in oyster homogenates for total and fecal e. coli and total vibrionaceae ; however , the levels of bacteria were generally 10- to 100-fold less than in filter - feeding molluscan shellfish , which bioconcentrate bacteria from the seawater within their edible tissues . by definition , fecal coliforms , including fecal e. coli , are gram - negative bacteria which ferment lactose with the production of acid and gas at 44.545.5c . such organisms are indicative of human gut bacteria and can be used as indicators for the possible presence of human fecal pathogens in foods and water . the mpn procedure capitalizes on the use of lactose - containing media , the collection of gas , and growth at 44.545.5c , so that positive isolates meet the classical definition for fecal coliforms . mpn - mug procedures use mug as an indicator for the presence of e. coli , since e. coli are the primary producers of gud at these elevated temperatures ; therefore , positive mpn results in the presence of mug are considered confirmatory for the presence of e. coli . current mpn assays for total and fecal coliforms , or e. coli , are tedious , costly , and time consuming . the incorporation of mug into ec broth for the mpn detection of e. coli has hastened the analysis over previous mpn methods but is still complex and produces false positives which can compromise results . according to the u.s . food and drug administration 's bacteriological analytical manual , the mpn - mug assay requires the use of new borosilicate glass tubes as well as new gas collection ( durham ) tubes and the tubes must be prescreened for fluorescence , as some tubes exhibit autofluorescence . in our hands , autofluorescence was a problem and may have contributed to the weakly positive mpn - mug results for e. coli o14:nm , o42:h2 , o103:h2 , and o111:nm , which were consistently negative by the mug overlay procedure ( table 1 ) . in the united states , oysters and their growing waters are monitored for fecal coliforms or e. coli using a 5-tube mpn approach , which means that 1530 or more new tubes may be required for a single analysis , depending on the number of dilutions required and the number of presumptive positive tubes that need to be transferred from lauryl sulfate tryptose broth to ec broth , thus making this procedure costly and impractical for routine use . since one of the major obstacles in monitoring for environmental contamination and food safety is the lack of practical and cost effective assays , our goal was to produce simple , more rapid , and inexpensive procedures to enhance monitoring efforts . this paper provides new tools to monitor for total and fecal e. coli and total vibrionaceae levels . in this study , we developed a simple plate cultivation and mug - membrane overlay technique to detect bacteria that are likely fecal e. coli based on gud production and their ability to grow at 44.5c . for our overlay procedure for total e. coli , we recognize that occasional pathogens other than e. coli may be gud positive , like some salmonella and shigella strains ( table 2 ) , or some streptococci ; however , the detection of other occasional pathogens simply enhances the benefits of using this test as a potential regulatory tool . endogenous levels of gud , which are high in some fish and shellfish [ 8 , 17 ] , are not a problem in our plate - based membrane overlays , since the overnight incubation of the plates at either 37c or 44.5c degrades endogenous , tissue - specific enzymes well before the plate is overlaid with the membrane containing the fluorogenic substrate . consequently , in the many assays we performed on oysters , we saw no background fluorescence , even though oyster tissues are known to contain gud . in addition to the complexities of performing traditional mpn - based assays , analyses for vibrionaceae in seawater and shellfish are usually complex and are generally limited to picking a few isolates from a plate for complex biochemical or molecular biological testing for specific pathogenic strains . monitoring of shellfish or seawater for pathogenic vibrios has failed to stop outbreaks of v. parahaemolyticus , while routine monitoring programs for v. vulnificus are nonexistent in spite of the fact that v. vulnificus in oysters causes occasional illness and death . acceptable baseline or threshold levels of vibrionaceae and total or fecal e. coli should be determined for implementation into regulations for harvesting or distribution of shellfish . threshold levels currently exist for coliforms in harvesting area waters and shellfish meats [ 3 , 4 ] but are based on mpn approaches . no methods have been established to date for regulation of shellfish harvesting or distribution based on total vibrionaceae levels or e. coli levels using simple overlay methods . in screening 37 strains of non - o157:h7 e. coli that were associated with major foodborne outbreaks of illness , 24 ( 65% ) were positive by the mug overlay and 23 ( 62% ) were positive by the mpn - mug assay . in contrast , other studies showed the recovery of gud in 95.5% , 96.5% , and 91% of the isolates examined . potential reasons for these differences in gud distribution among the isolates were suggested by chang et al . , but there have been no conclusive studies explaining such variability . in addition to a subset of non - o157:h7 isolates that were mug negative , 43 isolates of e. coli o157:h7 that were derived from a wide variety of food - borne outbreaks were mug negative . the failure to detect o157:h7 strains is a limitation of both mpn - mug and the mug overlay methods . this finding does not lessen the importance of the mug membrane overlay procedure , since o157:h7 strains constitute a minority of the e. coli in environmental isolates and other mug - based cultural assays , like the u.s . food and drug administration - recommended mpn - mug assay for e. coli , also fail to detect the o157:h7 serotype . escherichia coli o157:h7 strains do not contain enzymatically active gud , although these strains contain the gusa ( uida ) gene , which encodes gud and two regulatory regions . there does not appear to be regulatory repression to restrict gud formation , since antibody against gud detects the enzyme ; however , the gud is in an inactive form lacking hydrolytic activity . the cause for this inactive protein is the presence of a guanosine - guanosine insertion causing a frameshift resulting in the introduction of a premature termination codon in the gusa gene in all strains of e. coli o157:h7 tested , but not in other serotypes . this mutation accounts for the presence of an incomplete protein lacking gud activity ; therefore , mug - based assays are not appropriate for the detection of o157:h7 isolates . advantages of the overlays are that they are far simpler , less expensive , and directly quantitative , unlike mpns which give statistical probabilities of counts . culturing bacteria on nonselective tsa - n also has an advantage over selective media , like mcconkey agar with or without mug , since selective media are often inhibitory to environmentally stressed bacteria . with multiplex overlays , we have consolidated assays to simplify monitoring in order to drive down costs for screening and to promote more frequent testing for total e. coli and vibrionaceae . both the mug overlay and the copp assays are relatively fast , with results derived within 24 hours . mpn assays require up to 3 days for completion ( 2448 hours incubation in lst broth followed by 24 hours in ec broth ) and additional time if e. coli confirmation via biochemical testing is desired . together , the mug and copp overlay methods provide new options for quantifying total and fecal e. coli and vibrionaceae in seawater , shellfish , and other food and environmental matrices . unlike the original copp assay , which used cellulose acetate membranes that could be prepared in advance , dried for storage , and soaked in buffer before overlay [ 1 , 2 ] , the cellulose membranes used for copp and mug assays in this paper can not be prepared in advance or dried . this is because copp substrate covalently bonds to cellulose acetate membranes , but not to plain cellulose membranes , and mug does not bind to either membrane . although we used cellulose acetate and plain cellulose membranes to screen stock cultures for lysyl aminopeptidase activity , we used cellulose membranes exclusively for multiplex assay for total coliforms and total vibrionaceae . this was because copp assays on cellulose acetate membranes produced a bright light blue fluorescence while mug produced a stronger blue fluorescence ; thus , differentiation of the two colors was difficult at times . it became clear that the green and blue fluorescence obtained on the plain cellulose membranes was easier to discriminate between than blue and light blue fluorescence on the cellulose acetate membranes . multiplex overlays for the simultaneous detection of both total e. coli and total vibrionaceae may be performed when both groups of bacteria are in relatively the same proportion ; that is , both can be enumerated from the same dilution plate . methods to monitor molluscan shellfish harvesting and distribution , much like methods for other foods , water , or environmental samples , are often outdated and out of touch with today 's needs . it is necessary to shift analytical and regulatory paradigms to simpler , more practical , and cost effective measures to monitor relative levels of contamination . given the development of simple membrane overlay assays , we propose the establishment of membrane overlay - based monitoring for total and fecal e. coli and total vibrionaceae . for routine monitoring purposes , it may be time to shift from a search for specific vibrio pathogens to the enumeration of total vibrionaceae . environmental factors responsible for increases in human pathogenic vibrios within shellfish or their growing waters will likely cause an increase in the levels of many other vibrionaceae family members , including pathogenic strains ; therefore , the copp assay could prove useful in regulating shellfish harvesting based on total vibrionaceae counts . european standards for shellfish harvesting , where coliform levels are currently used to regulate shellfish harvesting and distribution [ 3 , 4 ] . in an aquaculture setting , the simple copp assay may serve as an index of fish health and disease , where spikes in vibrionaceae levels over what is normal and customary would suggest a need for more refined assays for specific pathogens and for corrective actions to be implemented within the aquaculture or hatchery setting . membrane overlays are simple and practical for routine monitoring and could enhance food safety by allowing increased numbers of samples to be tested at a reduced cost or by providing a simple alternative to procedures that are too rigorous for routine use .

kummell 's disease is a spinal disorder characterized by delayed post - traumatic collapse of a vertebral body with avascular necrosis . although definitive treatment for kummell 's disease has not been established , it has been reported that percutaneous vertebroplasty or kyphoplasty has shown good results . however , these procedures are not recommended for severely collapsed vertebral bodies because of the risk of cement leakage or technical difficulties . authors report a rare case of spontaneous reduction in vertebral height by the insertion of a working cannula into the vertebral body in kummell 's disease .

a 73-year old female patient 150 cm tall and weighing 50 kg visited our hospital presenting with the complaints of lower back pain and radiating pain to right lower extremity . at first , mild lower back pain had occurred from time to time , but severe pain with a visual analogue score ( vas ) of 8 or higher started to occur three months prior to her visit . the patient did not have any particular medical history except osteoporosis diagnosed in her late fifties . she felt pain as if her body was collapsing or sinking , accompanied by pulling pain at the hip and groin . the pain became more severe when standing up and moving , and relieved when lying down at rest . the physical examination showed tenderness at the first lumbar ( l1 ) spinous process region . the plain radiograph showed severe osteoporosis over the vertebral bodies , a severe compression fracture of the l1 vertebral body in which the anterior compression rate was 70% or higher , and an intravertebral vacuum cleft in the vertebral body ( fig . 1 ) . magnetic resonance imaging showed a low signal area of l1 in the t1 weighted image and a high signal in the t2 weighted image ( fig . 2 ) . despite conservative treatment for more than four weeks including continuous epidural pain control , the patient 's pain did not decrease and she was rejecting surgical procedures such as a spinal fusion . although the case was not a general indication of percutaneous vertebroplasty and kyphoplasty due to the severe collapse of the vertebral body , we decided to do transpedicular kyphoplasty using this method to block the cement outflow by slowly injecting highly viscous bone cement . before the procedure , we had the patient take the prone position , supporting her abdomen with a pillow , in order to reduce abdominal lordosis . to check the patient 's state during the procedure , we monitored the blood pressure , heart rate , and oxygen saturation , with supplement of oxygen through a nasal cannula . for the sake of sedation and analgesia during the procedure , when needed , we injected fentanyl intravenously with a total of 100 g . using the c - arm image , we verified the pedicle of l1 and determined a working cannula implantation position and angle . local infiltration anesthesia was performed with 1% lidocaine on the expected cannula implantation pathway . under fluoroscopic c - arm guidance , we implanted the cannula into the fractured vertebral body through the vertebral pedicle . at the moment when the left cannula was inserted into the vertebral body , air flowed into the vertebral body with a popping sound , and the level of the vertebral body recovered spontaneously ( fig . the cannula was also inserted into the right side , and balloons were inflated with a pressure of 50 - 100 psi to recover the level of vertebral body . then , 4.5 ml of bone cement was slowly injected through each side of cannulas through the l1 vertebral body ( fig . 4 ) . while injecting the bone cement , cement leakage out of the vertebral body after the procedure , the vas of the patient was 2 , indicating that the preoperative pain almost disappeared . currently , the patient during follow - up visits has not received any particular treatments except for the medication for osteoporosis . kummell 's disease is a delayed disorder characterized by avascular necrosis and the collapse of a vertebral body . the avascular necrosis accumulates gas or liquid inside the vertebral body , which is characteristically found as an intravertebral vacuum cleft on the computed tomography or plain radiograph . in an intravertebral vacuum cleft , a space formed by the necrotic bones , fusion of the fractured bones is difficult , and pseudoarthrosis , which could be moved by the patient 's posture or respiration , forms previous reports in the literature have mainly advocated conservative treatments such as bed rest and the wearing of aids as the treatment for kummell 's disease . however , the effect of the conservative treatment is often limited because vertebral body necrosis continuously progresses and natural healing hardly occurs in this disease . in the case where there is neurological damage , however , spinal fusion through general anesthesia is also quite limited because most of the patients are old accompanied by severe osteoporosis . bone cement injected during these treatments appropriately agglutinates fractures and resolves the instability due to the pseudoarthrosis by filling the vacuum cleft . in particular , kyphoplasty is considered as the more safe and effective procedure than percutaneous vertebroplasty in treating kummell 's disease because it can recover the vertebral body and reduce the cement outflow toward the space outside the vertebral body . although it is known that there is no absolute contraindication for these procedures as a treatment for vertebral compression fractures ; however , a severely collapsed vertebral body such as vertebra planar is generally considered as a contraindication for these procedures due to the difficulties in the procedure and the risk of cement outflow . in this case , we performed kyphoplasty without any other alternatives , although the patient had a severe osteonecrotic vertebral compression fracture in which the vertebral compression rate was higher than 70% and difficulties including the difficulties of the procedure itself as well as the risk of cement outflow were anticipated . unexpectedly , we experienced during the procedure spontaneous recovery of the vertebral body level as air suddenly flowed into the vacuum cleft of the vertebral body . this was rare even for us , and no similar cases were found in the literature . the possibility of an air embolism concerned us since air suddenly flowed into the vacuum cleft of the vertebral body ; however , no complications including an air embolism occurred due to the flow of air into the vertebral body during the recovery process for the vertebral body . we assumed that the spontaneous recovery of the vertebral body level took place as the vacuum cleft was filled with air immediately after the cannula was inserted because negative pressure existed in the vacuum cleft where the intravertebral pseudoarthrosis had formed . although there was the risk of bone cement outflow since the vertebral body could be damaged due to the sudden recovery , the kyphoplasty was successfully performed by slowly and meticulously injecting the bone cement , which had a consistency that was more viscous than the normal bone cement . the patient achieved drastic analgesia and recovery of mobility after the procedure . in conclusion , a kyphoplasty is worthy of considering as a good treatment modality that may provide analgesia and recovery of mobility without any complications for patients with kummell 's disease accompanied by severe vertebral body compression fracture . in some cases where a vacuum cleft in which negative pressure exists has been formed , spontaneous recovery of vertebral body level could occur .

chinese propolis ( cp ) , an important hive product , can alleviate inflammatory responses . however , little is known regarding the potential of propolis treatment for mastitis control . to investigate the anti - inflammatory effects of cp on bovine mammary epithelial cells ( mac - t ) , we used a range of pathogens to induce cellular inflammatory damage . cell viability was determined and expressions of inflammatory / antioxidant genes were measured . using a cell - based reporter assay system , we evaluated cp and its primary constituents on the nf-b and nrf2-are transcription activation . mac - t cells treated with bacterial endotoxin ( lipopolysaccharide , lps ) , heat - inactivated escherichia coli , and staphylococcus aureus exhibited significant decreases in cell viability while tnf- and lipoteichoic acid ( lta ) did not . pretreatment with cp prevented losses in cell viability associated with the addition of killed bacteria or bacterial endotoxins . there were also corresponding decreases in expressions of proinflammatory il-6 and tnf- mrna . compared with the mastitis challenged cells , enhanced expressions of antioxidant genes ho-1 , txnrd-1 , and gclm were observed in cp - treated cells . cp and its polyphenolic active components ( primarily caffeic acid phenethyl ester and quercetin ) had strong inhibitive effects against nf-b activation and increased the transcriptional activity of nrf2-are . these findings suggest that propolis may be valuable in the control of bovine mastitis .

inflammation of the mammary gland ( mastitis ) caused by invading pathogens is common among lactating dairy cows and leads to considerable economic loss through reduced milk yield , impaired milk quality , and increased veterinary costs . globally , mastitis is one of the most costly bovine diseases for the dairy industry , estimated to cost approximately $ 1.8 billion annually in the united states . the differences in these types of mastitis are based on the invading pathogen(s ) and resolution of the infection . clinical mastitis is stronger and more acute than the subclinical mastitis , and it is caused mainly by gram - negative bacteria such as escherichia coli . the subclinical mastitis elicits milder inflammation and persists over the life span of infected cows . gram - positive bacteria , like staphylococcus aureus , are involved with the subclinical mastitis infection . endotoxin ( lipopolysaccharide , lps ) and lipoteichoic acid ( lta ) are considered as the main virulence factor of e. coli and s. aureus , respectively [ 3 , 4 ] . moreover , proinflammatory cytokine tumor necrosis factor- ( tnf- ) elicits both local and systemic inflammatory reactions and triggers the inflammatory cascade acute phase response in inflammation and participated in the acute phase of coliform mastitis [ 5 , 6 ] . to suppress proliferation of invading pathogenic mastitis - causing bacteria , modern dairy practice employs several strategies , including teat disinfection , antibiotic therapy , and culling of persistently infected cows . despite the great effectiveness of antibiotics , their use is coming under increasing public scrutiny due to the possible development of resistant pathogens ( like methicillin - resistant s. aureus , mrsa ) and food safety concerns ( like antibiotic residues in milk ) . considering the risks associated with antibiotic therapies for bovine mastitis , development of alternative treatment strategies for management of clinical and subclinical mastitis propolis is a resinous substance collected by honeybees ( apis mellifera l. ) from various polyphenol - rich plants . it has been used widely in folk medicine since ancient times and has attracted much attention in recent years for its various biological properties . in our recent studies , we determined that propolis had potent anti - inflammatory effects in macrophages and boosted cellular antioxidant defence systems [ 11 , 12 ] . previous literature has shown that propolis could inhibit the growth of several different bacterial strains known to cause mastitis , as well as some antibiotic - resistant s. aureus strains [ 1315 ] . nevertheless , little is known about the effects of propolis on mastitis responses in bovine mammary epithelial cells ( bmecs ) . in the present study , we studied the impact of the effect propolis when bovine mammary epithelial cells were challenged with heat - killed mastitis - causing bacterial cells , as well as selected agents also associated with tissue response to mastitis . lps ( escherichia coli 0111:b4 ) , lta ( from staphylococcus aureus ) , caffeic acid , caffeic acid phenethyl ester ( cape ) , chrysin , ferulic acid , galangin , kaempferol , pinocembrin , and quercetin were purchased from sigma ( st . louis , mo , usa ) . high performance liquid chromatography- ( hplc- ) grade methanol was obtained from merck ( darmstadt , germany ) . recombinant human tnf- was purchased from peprotech ( rocky hill , nj , usa ) . culture plates were obtained from coring life science ( lowell , ca , usa . ) . the pi / rnase staining buffer kit , fitc - conjugated annexin v , and binding buffer were obtained from bd biosciences ( san diego , ca , usa ) . other chemicals were of analytical grade and purchased from sangon biotechnology ( shanghai , china ) . chinese propolis ( cp ) was obtained from colonies of honeybees , a. mellifera l. , in shandong province in the summer of 2010 , and the main plant origin was poplar ( populus spp . ) . briefly , raw propolis ( 100 g ) was extracted by 95% ( v / v ) ethanol ( 1 l ) and sonicated at 40c for 3 h. the supernatant was collected and filtered to remove the residues . the raw propolis was extracted for three times . then the supernatants were collected and evaporated in a rotary evaporator under a reduced pressure at 50c to evaporate the ethanol . dried ppe were stored at 20c until further use . for the in vitro studies , cp was redissolved directly in ethanol to a concentration of 20 mg / ml and sterilized using a 0.22 m syringe filter ( pall , port washington , ny , usa ) . the final concentration of ethanol in the cell culture was less than 0.5% ( v / v ) . bovine mec line mac - t cells were generously provided by professor fengqi zhao ( university of vermont , burlington , usa ) which were cultured in high - glucose dulbecco 's modified eagle 's medium ( dmem , hyclone , fremont , ca , usa ) supplemented with 100 u / ml of penicillin , 100 g / ml streptomycin , and 10% ( v / v ) heat - inactivated fetal bovine serum ( fbs , gibco , carlsbad , ca , usa ) at 37c and 5% co2 in a humidified incubator . we challenged these cells with heat - inactivated bacteria particles of the mastitis - causing pathogens e. coli strain 1303 and s. aureus newbould 305 . details regarding the culture of e. coli or s. aureus pathogens and usages of these heat - inactivated bacteria particles to challenge bmecs were described previously . e. coli and s. aureus strains were grown ( 37c ) in lysogeny broth ( lb ) medium to the logarithmic phase of culture growth . heat inactivation was performed in an 80c water bath for 1 h to kill all live cells and verified through control plating . subsequently , cells were spun down , washed twice with pbs , and later then resuspended with dmem at a density of 5 10 cells / ml . aliquots were stored frozen at 20c until used . cell viability assay was performed using the cck-8 kit ( dojido , kumamoto , japan ) according to the manufacture 's instruction . briefly , 10 10/ml mac - t cells were seeded into 96-well culture plates . after 24 h incubation , cells in each well with specific treatment were incubated with 10 l of cck-8 at 37c for 2 h before measuring the od at 450 nm with a microplate reader ( spectramax m5 , molecular devices , sunnvale , ca , usa ) . cells were stained with annexin v - fitc and pi and then evaluated for apoptosis by flow cytometry according to the manufacturer 's protocol ( bd biosciences , san diego , ca , usa ) . briefly , 10 cells were washed twice with pbs and stained with 5 l of annexin v and 5 l of pi ( 50 g / ml ) in washing buffer from the kits for 15 min at room temperature in the dark . apoptotic cells were counted in the lower right quadrant corresponding to early apoptotic cells ( annexin v - positive ) and those in the upper right quadrant corresponded to late apoptotic cells ( annexin v- and pi - positive ) . total rna was extracted with the rna pure kit ( aidlab biotechnologies co. , ltd . , reverse transcription was done using the primescript rt reagent kit ( takara , dalian , china ) with 1 g total rna . quantitative real - time pcr was carried out with sybr premix ex taq ( takara ) following the manufacturer 's instructions with 1 : 10 diluted cdna template and using a standard two - step reaction . primers for target genes that covered introns were designed with the primer5 software ( premier biosoft international , palo alto , ca ) and listed in table 1 . after overnight incubation , 30 ng of firefly luciferase reporter plasmid pgl4.2-nf-b - luc and pgl4.37-luc2p / are / hygro vector ( promega , madison , wi , usa ) were transfected to drive transcription of nf-b and are , respectively , by using lipofectamine 2000 ( invitrogen , carlsbad , ca , usa ) . sea pansy luciferase reporter plasmid ( prl - tk , 5 ng ) was transfected to normalize the transfection efficiency . the pcdna3.1 empty vector was used to compensate the total expression plasmids to 500 ng / well . luciferase activities were measured 24 h after specific treatments by using the dual - glo luciferase assay system ( promega ) . data are expressed as the means sd from at least three independently performed experiments . one - way analysis of variance ( anova ) followed by student - newman - keuls ( snk ) multiple - comparison test was used to determine statistical significance for multiple comparisons , and student 's t - test was used for comparisons of two groups . we analyzed the major polyphenolic compounds in chinese propolis using our previously developed hplc method . the major polyphenolic components were chrysin , pinocembrin , pinobanksin , galangin , and cape . as shown in figure 1(a ) , not all of these stimuli caused cell viability decreases in mac - t cells . only lps and heat - killed e. coli and s. aureus , but not tnf- and lta stimulation , lead to significant cell viability losses ( 15% to 52% , p = 0.0009 , 0.0041 , and 0.001 for lps , e. coli , and s. aureus , resp . ) . heat - killed bacterial strains caused more serious cell viability losses than lps . to test the effects of cp on protecting against the cell viability decreases caused by mastitis pathogens , various concentrations of cp also shown in figure 1(a ) , tested concentrations of cp ( 5 , 10 , and 15 g / ml ) pretreatment were safe to mac - t cells . cp pretreatment ( 10 and 15 g / ml ) significantly mitigated cell viability loss by lps ( p < 0.05 ) and heat - killed e. coli ( p < 0.05 ) . s. aureus - induced cell viability losses can be inhibited by 15 g / ml cp pretreatment ( p < 0.05 ) . cell apoptosis analysis by flow cytometry showed that 10 particles / ml heat - killed mastitis strains caused a substantially increased percentage of apoptotic cells , but none of the other stimuli , tnf- , lps , or lta , cause any changes on apoptotic cell numbers ( figure 1(b ) ) . nevertheless , pretreatment with 15 g / ml cp in mac - t cells showed a significantly lower number of apoptotic cells ( p < 0.05 ) compared to heat - killed e. coli- and s. aureus - treated cells , which was consistent with the above result obtained from cck-8 cell viability assay . as shown in figure 2 , all of these mastitis pathogens , except for lta , lead to significant increases of il-6 and il-8 ( p < 0.05 ) . lta and heat - killed s. aureus stimulation also failed to induce expression of tnf- compared with uninfected cells ( p > 0.05 ) . on the contrary , heat - killed e. cp - pretreated mac - t cells have much slighter changes of il-6 and tnf- mrna compared to their corresponding stimuli controls . surprisingly , cp lead to ~8-fold changes of il-8 , significantly higher than tnf- , lps , and heat - killed e. coli stimuli ( p < 0.05 ) . as shown in figure 3(c ) , only gclm expression was affected by several mastitis pathogens ( tnf- and two heat - killed mastitis strain particles ) . we noticed that cp treatment in mac - t cells leads to substantial increases in ho-1 , txnrd-1 , and gclm expressions ( p < 0.05 ) . ho-1 expressions were further elevated in cp - pretreated mac - t cells undergoing mastitis challenges ( figure 3(a ) ) . upregulated txnrd-1 was unchanged in cp - treated mac - t cells ( figure 3(b ) , p < 0.05 ) , regardless of these different mastitis pathogens . furthermore , cp treatment itself can not induce expression of gclm but it substantially increased in mastitis pathogen challenged cells along with cp . these results indicated that cp could elicit the antioxidant defense system in the bmecs cells undergoing mastitis challenges . as shown in figure 4 , the increased expressions of proinflammatory cytokines il-6 ( figure 4(a ) ) and tnf- ( figure 4(c ) ) were suppressed by cp in a time- and dose - dependent manner . as for another important chemokine , il-8 , its expression was significantly driven by tnf- and immediately peaked at 3 h after tnf stimulus ( figure 4(b ) ) . cp treatment strongly promoted il-8 expression , which reached its peak after 9 h tnf stimulus . we also found that tnf- had very limited effects on antioxidant gene expressions , ho-1 , txrnd , and gclm ( figures 4(d)4(f ) ) . to determine the effects of cp as well as its purified compounds on nf-b and are transcriptional activity , luciferase reporter assays were applied in hek-293 t cells . as shown in figure 5 , nf-b activity was significantly decreased by approximately 70% ( p < 0.001 ) . the luciferase activity derived from the are promoter was consistently increased by approximately 4.7-fold after treating with 20 g / ml cp ( figure 5(b ) , p < 0.001 ) . moreover , significant decreases in nf-b activity were found in cells treated with cape ( 5 m , 45% , p < 0.001 ) , quercetin ( 50 m , 73% , p < 0.001 ) caffeic acid ( 50 m , 12% , p < 0.05 ) , and ferulic acid ( 50 m , 11% , p < 0.05 ) ( figure 5(a ) ) . in parallel , the luciferase activity derived from the are promoter was consistently increased after treatment with cape ( 5 m , 2.5-fold , p < 0.001 ) , quercetin ( 50 m , 5.1-fold , p < 0.01 ) , kaempferol ( 50 m , 3.6-fold , p < 0.001 ) , and pinocembrin ( 50 m , 2.9-fold , p < 0.01 ) . regardless of increased pressures of cutting down the antibiotic usage in the husbandry in the european union and us , mastitis treatment is continued after the initial usage of antibiotics , mainly for chronically occurring subclinical mastitis . the increasing scientific data on the benefit of natural products encourages us to explore novel nonantibiotic agents for mastitis therapies . in this study , the potential utilization of chinese propolis as an anti - inflammatory reagent and immune modulator against bovine mastitis was examined in bmecs . previous studies have demonstrated that propolis has great antimicrobial properties against bacterial fungi , protozoan , and even yeast pathogens . despite chemical variations of propolis from different geographic origins / plant sources , the antibacterial activity by propolis always exists . in accordance with published literature , chinese propolis extracts on gram - positive s. aureus cells appear to be bactericidal but show only limited activity against gram - negative e. coli bacteria . based on the literature , we applied 10 particles / ml of the heat - inactivated bacteria to the bmecs , which is approximately 30 particles challenged per host cell [ 22 , 23 ] . this approach represented a reliable robust stimulus model considering that 10 to 10 cfu of bacteria particles per ml as peak values in the milk were produced from cows after mastitis infection . also , we confirmed that propolis ' bactericidal effects are potent in controlling mastitis - causing s. aureus strains , which are most frequent but more difficult to eradicate by conventional antibiotic therapies . it has been known that acute coliform mastitis is caused by endotoxin - producing acute coliform bacteria ( specifically , e.g. , e. coli , enterobacter , and klebsiella spp . ) and is characterized by a rapid and intense increase in the somatic cell count ( scc ) , while subclinical mastitis caused by s. aureus is characterized by a more moderate and delayed scc increase that leads to chronic and , in some circumstances , life - long infection . mammary tissue damages induced by mastitis pathogens also have been shown to be linked with increased scc and mainly induced by either apoptotic or necrotic mammary cells . we explored and compared the effects of major different various mastitis pathogens on bmecs cell viability and cell apoptotic features . we found that tnf- and lta stimulation did not cause any significant losses on the cell viability or cell apoptosis to bmecs , coinciding with other in vitro studies using bovine mammary cells [ 1 , 5 ] . meanwhile , previous findings on proapoptotic effects by heat - killed e. coli and s. aureus were inhibited by cp pretreatment , suggesting that cp has modulating effects on the cellular apoptosis cascade , such as blocking the activation of caspases [ 1 , 26 ] . we noticed that the e. coli cell wall component lps only decreased cell viability but did not induce cell apoptosis , suggesting that the influences of bacterial cell wall components on bmecs may be different from those mastitis bacteria strains . moreover , as the internalization process of bacteria pathogens to the cells differs among bacteria strains , the different signal transduction mechanisms requiring the interaction between cp and cell wall components as well as the host cells are in need to be further clarified . bovine mastitis is initiated by the entry of bacteria through the teat canal . shortly after entry of the invading pathogen , the resident leukocytes together with epithelial cells initiate the inflammatory response necessary to eliminate the invading bacteria . different mediators of inflammation , especially some inflammatory cytokines , which are expressed during this stage will participate in the innate immune defense to respond to the earliest stages of infection after mastitis pathogen / stimulus exposure . il-6 and tnf- are the two most predominant proinflammatory cytokines during the acute phase responses of mastitis . in heat - inactivated e. coli challenged bmecs , significant increases of il-6 and tnf- gene expression were observed ( figure 2 ) , a finding consistent with a previous study using a different mastitis strain of e. coli . compared to lps and tnf- , heat - inactivated e. coli elicited stronger inductive effects on these inflammatory cytokines expressions , suggesting that some other virulence factors from e. coli ( like lipoproteins or flagellum ) may be involved in the innate immune defense of the bovine epithelium cells [ 23 , 28 ] . in contrast to e. coli challenged models , lower levels of inflammatory response were found after s. aureus compared with e. coli , which has been found by previous studies . in these studies no increases in the concentrations of tnf- , il-1 , and il-8 in milk were found after intramammary challenge by s. aureus . in our experiments , we did not notice any significant changes on these inflammatory cytokine genes following s. aureus or lta stimulation . previous reports have suggested that s. aureus mastitis is characterized by a more moderate and delayed inflammatory responses and limited cytokine responses [ 22 , 25 ] . expanded time - course studies of cp effects by s. aureus and lta stimulation need to be conducted . propolis has been used historically as a folk medicine since ancient ages times and has since been safely used as a substitute medicine or supplement to improve human health in modern days . although the effective components in propolis vary significantly according to the mother plant that the honeybees collect it from , the bioactivities of propolis are always present . our previous studies have confirmed the anti - inflammatory effects of chinese propolis ( poplar type ) by protecting murine macrophages from lps challenges and controlling proinflammatory cytokine releases in lps - induced mice endotoxemia . similar to our previously published results , the proinflammatory cytokine expressions ( il-6 and tnf- ) were inhibited by cp treatment following coliform mastitis pathogen challenges ( heat - inactivated e. coli as well as endotoxin ) . besides , through our comprehensive tnf- stimulation model , we found that cp effectively inhibits the expression of these two genes in a time- and dose - dependent manner . through our previous studies using lps challenged murine macrophages models , the inhibition on nf-b activation by cp may be the source of its anti - inflammatory effects . we knew that polyphenolic compounds are among the most prominent components of propolis because they are considered responsible for most of its properties . in parallel with previous studies , cape , quercetin , and caffeic it should be noted that in this study , the expression of il-8 ( also known as cxcl8 ) increased in cp - treated cells in several different stimulus models . various animal models of inflammation have demonstrated il-8 as a principal chemotactic factor directing neutrophil recruitment to activation at the inflammatory focus . expression of il-8 is known to be controlled by multiple transcriptional factors ( activator protein , ap , -1 , and nf-b and nf-b repressing factor , nrf ) and involved with some coactivator cbp/300 complexes . it also has been shown that the pivotal importance of c / ebp to activate il-8 expression in mecs is independent of nf-b activation . therefore , in this study , as we have found that the nf-b transcriptional activity was inhibited by cp and its bioactive compounds , we assumed that other transcriptional factors , like c / ebp , which are possibly activated by cp , may contribute to the enhancement of il-8 expression in mastitis pathogens challenged mec . it is well - known that multiple antioxidant genes may be induced by diverse plant - derived natural products . these antioxidant genes , such as ho-1 , txnrd1 , gclm , sod , and nqo-1 , encode several detoxifying ( phase ii ) enzymes and endogenous antioxidants to provide protections against external stresses , including oxidative stress and inflammation . in particular , nrf2 has been shown to be involved in the inductions of phase ii detoxifying enzymes and antioxidants by natural polyphenols in bmecs suffered from oxidative stress . ho-1 is well - known to be a major antioxidant enzyme that plays an important role in the antioxidant defense against inflammatory stresses in different cell types , such as macrophages , keratinocytes , and epithelial cells [ 38 , 39 ] . thioredoxin reductase ( trxr ) , which catalyzes the reduction of the active site disulfide of thioredoxin ( trx ) , is an nadph - dependent homodimeric oxidoreductase . as trxr is an antioxidant enzyme that can scavenge ros , cp - induced trxr1 expression may indicate the activation of cellular defense systems against tnf and other mastitis pathogen challenges . our recent study found that endogenous antioxidant defenses systems could be activated by chinese propolis in murine macrophages . in parallel with this study , these increased antioxidant gene expressions are in keeping with cp 's inductive effects of nrf2-are transcription activity . furthermore , several specific active polyphenolic compounds from propolis have been shown to activate nrf2-are signaling , including cape , kaempferol , quercetin , and pinocembrin . our studies have demonstrated that chinese propolis has strong protective effects against various mastitis pathogen insults to bmecs . apart from cp 's bactericidal effects , it clearly prevented cell viability losses / apoptosis induced by lps and heat - inactivated mastitis strains ( e. coli and s. aureus ) in bmecs . these effects were partly achieved by modulating expression of inflammatory cytokine genes and boosting antioxidant defense genes . the possible mechanisms for cp against bovine mastitis may be attributed to its abundant polyphenolic components ( mainly cape and quercetin ) , which strongly inhibited nf-b transcription activity and increased the transcriptional activity of the nrf2-are pathway . field studies are needed to confirm that propolis may be developed as nonantibiotic therapy strategy for the control of cow mastitis .

[ purpose ] the aim of this study was to ascertain the optimum strategy for implementing a physical activity intervention in patients on hemodialysis by investigating the physical characteristics of elderly patients on hemodialysis , and their attitude to physical activity and level of daily activity . [ subjects ] the subject were 10 elderly patients on hemodialysis . [ methods ] they wore a physical activity monitor for 1 week . data obtained were analyzed for hemodialysis and non- hemodialysis days , and two - way analysis of variance was used to compare the number of steps and activity levels . a questionnaire was administered to investigate the stage of psychological preparedness for exercise and attitudes toward / awareness of exercise . [ results ] there was no significant difference in the number of steps or exercise levels on hemodialysis and non- hemodialysis days . however , on both types of days , subjects spent long periods not engaged in any activity . most of their activity was either inactivity or sedentary behavior . [ conclusion ] patients on hemodialysis with low physical activity levels are considered to have poor physical function and exercise tolerance . to maintain and improve the physical function of patients on hemodialysis , it will be necessary to reduce their time spent in inactive , and comprehensive care that covers psychosocial aspects should be provided to promote the proactive improvement of physical activity and their attitudes to exercise .

exercise tolerance generally decreases in patients on hemodialysis ( hd ) who have diminished musculoskeletal muscle strength and muscle atrophy , reduced muscle blood flow distribution , a decline in cardiopulmonary function , and severely reduced activities of daily living . previous studies have found that a low level of physical activity ( pa ) affects physical function , and it has psychological effects that influence the prognosis for survival1,2,3,4 . recently , exercise intervention and high levels of pa have been recommended for patients on hd . studies have found that these are effective for improving exercise tolerance ( i.e. , an increased maximum oxygen uptake and anaerobic threshold ) , pulmonary function ( i.e. , a lower ventilatory volume at a given submaximal load intensity ) , cardiac function ( i.e. , a lower heart rate at a given submaximal load intensity ) , coronary risk factors ( e.g. , a lower systolic blood pressure , higher high - density lipoprotein cholesterol level , and lower neutral fat level ) , and survival prognosis and for preventing the progression of coronary artery stenotic lesions1 , 5,6,7,8,9,10,11 . although a high level of physical exercise exerts a wide range of beneficial effects , it has so far been difficult to improve the low level of pa in patients on hd . patients with low levels of pa tend to be elderly women who possess factors that are strongly correlated with diabetes and atherosclerosis9 . particularly , helping such patients maintain a high level of pa is an issue given their level of physical function , and it requires an approach that takes activity patterns , psychological factors , and environmental adjustments into account . in recent years , many elderly patients above 60 years old have started to undergo dialysis treatment above 60 years old , or they have been undergoing dialysis treatment , in addition to those who for a long time ( since a young age ) . as the number of elderly patients on hd increases , it is important to maintain appropriate pa to protect against decreases in physical function and activities of daily living function caused by disuse in daily life . however , in japan , rehabilitation is not targeted at patients on dialysis during medical treatment . many patients on dialysis do not readily participate in exercise classes at fitness clubs or communities because of dialysis treatment ; consequently , they miss the opportunity to exercise or receive instruction . as there is a lack of a good exercise environment for elderly patients on hd , a large amount of motivation is required to engage such patients in pa . moreover , elderly patients on hd tend to have low participation in social activity , and they mostly stay indoors , which further reduces their physical levels of pa . in elderly patients on hd , the amount and intensity of pa are important . many previous studies , however , have focused solely on the amount of pa in terms of the number of steps taken by patients on hd per day , and few have addressed the level of everyday activities that patients on hd are engaged in or their attitudes toward exercise and their state of psychological preparedness . assessing the activity patterns of patients on hd with a focus on their pa levels and determining their attitudes toward exercise may enable the establishment of support methods that are best suited to raising their levels of activity . the current study aimed to identify the characteristics of elderly patients on hd and their attitudes toward exercise and to ascertain their everyday activity levels by using a pa monitor . the protocol for the research project conformed to the provisions of the declaration of helsinki ( as revised in tokyo in 2004 ) . a researcher described the research purposes , methods , risks , and benefits to the participants . then consent was obtained for the research study , and a questionnaire to collect personal information was administered to the subjects . subjects were recruited from amongst outpatients who visited two institutions in japan : a hospital and clinic that specialize in hd treatment . the inclusion criteria were age 60 years , 4 h of hd 3 times / wk renal disease , and hd for > 12 wk . to measure pa , subjects wore an accelerometer ( kenz lifecorder ex 4s version ; measurement interval ) to evaluate the amount of pa they performed for 1 wk , and their number of steps and level of exercise were calculated . they were obligated to wear the device at all times other than in the bath . the imported values for exercise level obtained from the accelerometer included periods in which no activity was recorded , and these periods treated as periods of inactivity . the levels of activity were as follows : pa intensity < 1 , sedentary behavior ( sb ) ; 13 , light - intensity activity ( lpa ) ; 45 , moderate - intensity activity ( mpa ) ; and 69 , vigorous - intensity activity ( vpa ) . the survey on attitudes toward exercise covered the subjects psychological state with respect to exercise based on the transtheoretical model10 ( a five - stage model comprising precontemplation , no interest at all ; contemplation , interested but not yet taking action ; preparation , engaging in exercise on an irregular basis ; practice , having engaged in continued regular exercise for < 6 months ; and maintenance , having engaged in regular exercise for at least 6 months and acquired the habit of exercising ) . items related to their exercise habits ( whether they engaged in habitual exercise and the type , frequency , and period for which it was continued ) and motivation for exercise ( assessed on a four - point scale of agree , somewhat agree , somewhat disagree , and strongly disagree ) and items concerning awareness of the need for and the effects of exercise , negative attitudes toward exercise , previous experience of exercise , and wish for instruction about exercise by medical staff ( yes / no answers ) were evaluated . regarding the analytical methods , accelerometer data were used to calculate the mean time per day spent at each level of pa ( inactivity , sb , lpa , mpa , and vpa ) . the number of steps and time spent at each level of exercise were also calculated separately for each subject on hd and non - hd days . two - way factorial analysis of variance ( anova ) was performed with hd / non - hd days and activity levels as independent variables and activity time as the dependent variable . the frequencies and percentages of the responses to survey on psychological state were calculated . the subjects were three men ( age , 68.7 2.3 years ; height , 163.7 1.2 cm ; weight , 58.8 5.0 kg ; and dialysis history : 4.6 2.1 years ) and seven women ( age , 65.3 6.6 year ; height , 152.7 3.7 cm ; weight , 52.4 11.6 kg ; and dialysis history , 2.1 1.3 years ) . the data of 8 subjects for whom no data were missing were used to calculate the pa levels as the main outcome of this study . in 2 cases with immeasurable levels , the accelerometer did not respond to the level of movement during walking even though the subjects were capable of walking independently . the number of steps was 2,805.1 1,742.0 on hd days and 4,717.0 2,991.6 on non - hd days ( p = 0.069 , effect size ( es ) = 0.64 ) , and the difference was not significant ( table 1table 1.physical activity intensity levels on the dialysis and non - dialysis daysdialysis daynon - dialysis daymeanmediansdmeanmediansdsteps ( steps)2,805.12,428.31,742.04,717.04,604.72,991.6inactivity ( min)792.5720.4315.8610.4599.4253.7sb ( min)557.8639.5282.3619.4811.5268.8lpa ( min)20.920.910.332.934.019.7mpa ( min)7.72.210.511.45.114.7vpa ( min)0.70.21.51.20.21.3sb : sedentary behavior ; lpa : light - intensity physical activity ; mpa : moderate - intensity physical activity ; vpa : vigorous - intensity physical activity ) . the mean number of steps was lower for hd elderly japanese patients than for non - hd elderly japanese patients ( men , 7,303 steps , and women , 6,705 steps ) . sb : sedentary behavior ; lpa : light - intensity physical activity ; mpa : moderate - intensity physical activity ; vpa : vigorous - intensity physical activity to verify whether there was a significant difference between exercise levels on hd and non - hd days , two - way factorial anova was performed with hd / non - hd days and activity levels as independent variables , and activity time as the dependent variable ( tables 1 and 2 ) . this showed a significant interaction ( f [ 4 , 28 ] = 6.0 , p < 0.001 , n = 0.46 ) . a test of the simple main effect for each level showed that it was not significant for hd / non - hd days ( f [ 1 , 70 ] = 0.72 , p = 0.79 , n = 0.01 ) . the simple main effect was significant for the activity level ( f [ 4 , 70 ] = 68.9 , p < 0.01 , n = 0.79 ) , and a multiple comparison test showed significant correlations between inactivity and lpa , mpa , and vpa and between sb and lpa , mpa , and vpa . however , there was no significant correlation between inactivity and sb , or between lpa , mpa , and vpa . the survey on attitudes toward exercise showed that 5 subjects ( 50.0% ) were in the precontemplation stage , 3 ( 30.0% ) were in the contemplation stage , and 2 ( 20.0% ) were in the practice stage and in the habit of engaging in regular exercise ( table 2table 2.results concerning psychological staten=10%ttm stageprecontemplation550.0contemplation330.0practice220.0ex habityes220.0no880.0habitual ex typegroup ex1walking1habitual ex frequency / wover 3 days110.02 days110.0none880.0habitual ex durationover 1 year110.016 months110.0less than 1 month880.0motivation for exagree550.0somewhat disagree550.0awareness of the need for exagree550.0somewhat agree550.0negative attitude toward exagree110.0somewhat agree660.0somewhat disagree220.0disagree110.0effect of exagree550.0somewhat550.0previous experience of exyes770.0no330.0ttm : transtheoretical model ; ex : exercise ) . motivation to exercise and awareness of the need to exercise was high in 5 subjects ( 50.0% ) and low in 5 ( 50.0% ) . seven subjects ( 70.0% ) disliked exercise and thought of it as too much trouble . the purpose of this study was to ascertain the characteristics of the pa levels and attitudes toward pa of elderly patients on hd . we found that their mean number of steps per day was lower than the reference value for non - hd elderly individuals . additionally , although there was no significant difference in the numbers of steps or activity levels on hd and non - hd days , the time spent in inactivity was long on both days , and most activity was sb . over 80.0% of the subjects were not in the habit of exercising , and although 70.0% had previous experience with exercising , their awareness of the need to exercise was now low ( 50.0% ) ; additionally , they tended to harbor negative feelings about exercising ( 70.0% ) . the decline in physical function seen in patients on hd was due to the effect of chronic renal failure and the fact that they were leading sedentary lifestyles , with low levels and intensities of exercise , which is also an important factor . in a previous study of pa , 795 ( 35.1% ) of among 2,264 patients on hd in the united states , a follow - up investigation of those patients 1 year later found that heart disease and peripheral complications were more frequent among those who had not engaged in exercise or pa , and the risk of death after 1 year was 1.62 times higher than that of those who did engage in exercise or pa . a 10 year follow - up study of patients who engaged in regular aquatic exercise and those who were inactive also found that mortality was higher for patients on hd13 . in terms of patterns of pa , there were no great differences in the average values on hd and non - hd days , but the absence of a difference in the number of steps may have been due to the wide standard deviation . in a previous study of pa on hd and non - hd days , majchrzak et al.12 , 14 , 15 showed that patients on hd had particularly low activity levels on hd days . they found that factors contributing to the decrease in pa on hd days included hd - induced fatigue and postural hypotension14 , 16 . patients on hd also spend about > 10 h / wk in treatment - associated rest , hemostasis after hd , and post - hd fatigue with a low blood pressure . focusing on the activity intensity patterns in this study , subjects spent the longest amount of time inactive , and even when they were active , the time spent in sb , which involved sitting or a similar activity , was extremely long on both hd and non - hd days . johansen et al.17 used accelerometry to measure activity levels in patients on hd over a 1-wk period and showed that patients on hd had significantly lower levels of activity than those of healthy individuals who were leading a sedentary lifestyle . guidelines in many countries , japan included , recommend that elderly people take at least 6,000 steps a day and engage in around 15 min of moderate pa , but patients on hd do not achieve this even on non - hd days . the length of these periods of inactivity and bed rest minimizes the effect of gravity , meaning that gravity - resisting muscles are not subjected to gravity for continued periods , resulting in inactivity . thus , they develop disuse muscle atrophy , which reduces muscle strength , generating a vicious cycle whereby declining physical function factors and pa exert a negative effect on each other15 , 16 , 18 , 19 . patients on hd are therefore recommended to be proactive in raising their levels of pa and incorporating exercise therapy . however , the question of the extent to which the pa of patients on hd can be increased is an important one . as shown by subjects in this study , patients on hd may have a low awareness of exercise , and even if they do regard it as necessary , they may not engage in it due to negative attitudes . a wide range of intervention methods are now being used to encourage pa by patients on hd , including intervention on non - hd days and before and during hd , as well as the use of home exercises . many patients on hd also have complications and may be worried about heart failure or hypertension immediately before hd and postural hypertension immediately after hd . thus , it is necessary to encourage pa that is best suited to each individual . counseling and efforts on the part of staff involved in hd to encourage patients on hd to increase their levels of regular physical exercise are also required5 . the present findings also suggested that patients on hd are looking for support from medical staff . according to bennett et al.6 , 20 , specialist staff and encouragement and commitment on the part of medical staff are factors that contribute to a successful continuous program . for medical staff , possessing the appropriate knowledge is also a factor that helps increase the level of physical exercise in patients on hd . improving the level of physical exercise has been shown to have a beneficial effect on mental health and physical function16 , 21 , and intervention through appropriate pa may provide the impetus for patients on hd to generate a virtuous circle . this study only included a small number of subjects , and thus we did not attempt to elucidate a causal relationship between pa and body strength characteristics . patients on hd with low levels of pa , however , reportedly also have poor physical function , exercise tolerance , and survival prognosis22 . in addition , this study focused on intensity rather than the number of steps . as may be understood from the results of this study , it is significantly difficult for patients to exercise on their own in the absence of exercise instructors due to their low motivation for exercise . in recent years , from the perspective of health - related behavioral science23 , however , emphasis has been placed on eliminating negative factors , such as reducing sitting time daily and changing patterns of inactive time ( i.e. , sitting behavior ) , rather than on positive factors for improving pa by promoting exercises , as was the case in this study . therefore , appropriate pa guidance is required , especially at medical institutions without stationed specialists to help with exercise . in the 2 cases with immeasurable pa levels in this study , the accelerometer did not respond to the levels of movement during walking even though the patients could walk independently . determining indexes of the amount of physical exertion for patients on dialysis is important . furthermore , a comprehensive care program that includes guidance on improving pa and the psychosocial aspects of exercise is needed .

injury to the spleen during routine colonoscopy is an extremely rare injury . diagnosis and management of the injury has evolved with technological advances and experience gained in the management of splenic injuries sustained in trauma . of the 37 reported cases of colonoscopic splenic injury , 12 had a history of prior surgery or a disease process suggesting the presence of adhesions . only 6 had noted difficulty during the procedure , and 31 patients experienced pain , shock , or hemoglobin drop as the indication of splenic injury . since 1989 , 21/24 ( 87.5% ) patients have been diagnosed initially using computed tomography or ultrasonography . overall , only 27.8% have retained their spleens . none have experienced as long a delay as our patient , nor have any had an attempt at percutaneous control of the injury . this report presents an unusual case of a rare complication of colonoscopy and the unsuccessful use of one nonoperative technique , and reviews the experience reported in the world literature , including current day management options .

endoscopic evaluation of the gastrointestinal tract offers both diagnostic and therapeutic options and has become the preferred procedure for the evaluation of gastrointestinal disorders . presented here are an illustrative example and a review of the world literature , with a focus on diagnostic and management options . a healthy , 75-year - old woman underwent screening colonoscopy at an outside facility and developed left upper quadrant abdominal pain over the ensuing days . cyst , and observational management was elected . over the next several months , the patient underwent serial ct scan examinations of her abdomen , which demonstrated a slowly but progressively enlarging splenic cyst . approximately 4 months after the inciting colonoscopy , the patient was referred to our facility for management . a 10x7-cm thin - walled splenic fluid collection was seen on ct ( figure 1 ) , abutting the abdominal wall and displacing the spleen medially . a fluid / fluid level could be seen within the collection , consistent with hematocrit effect ( blood cells separating from plasma and settling over time ) . the collection was percutaneously drained with an 8 french multi - holed catheter for 400cc of brown fluid , consistent with old blood . postprocedure ct confirmed complete collapse of the collection ( figure 2 ) and the catheter was removed . six days after the collection was drained , the patient presented with a return of her abdominal pain and was found on follow - up ct to have a recurrent 6x4-cm subcapsular splenic collection with a small hematocrit level , presumably from ongoing small hemorrhages . with the rapid reformation of the collection therefore , the patient was immunized with pneumococcal and hib vaccines and taken for elective splenectomy 2 days later . though laparoscopic splenectomy was considered , open resection was chosen given the patient 's age and likelihood of significant inflammatory changes . at laparotomy , she was noted to have a very mobile splenic flexure with an underdeveloped splenocolic ligament . inflammatory adhesions were found in the area of the spleen , and an 8x5-cm white cyst , slightly larger than the spleen itself , was found posterolateral to the spleen ( figures 3 and 4 ) . postoperatively , the patient did remarkably well and was discharged home 3 days later . presenting computed tomographic scan of patient with large undrained hemorrhagic splenic cyst abutting the left lateral abdominal wall . though barium enema has long been the gold standard for identification of colonic lesions , colonoscopy affords the ability to not only identify a lesion , but to provide tissue for pathologic examination and is often the definitive procedure for polyps . hence , colonoscopy is considered the accepted screening examination in all people over 50 years of age , and younger if a familial history of colon cancer is present . though overall very safe the 2 most common complications are hemorrhage following polypectomy ( range , 1.8% to 2.5% ) and perforation ( range , 0.34% to 2.14% ) . the injury was first reported in 1974 , with 2 patients sustaining hemorrhage , one of which resulted in splenectomy . since the first report , an additional 36 cases have been reported in the world literature , including the current case . the average age of the patients is 64.9 years ( range , 33 to 90 ) , reflecting the age for which colonoscopy is routinely indicated , with a preponderance of females ( 66.7% ) . difficult intubation of the colon may impart direct injury to the spleen during passage through the splenic flexure . dense adhesions between the colon and spleen from previous surgery or disease may result in tearing of the splenic capsule as the colonoscope is passed through the colon . telmos and others later added technical maneuvers to the list of risk factors , including slide - by , the alpha maneuver , straightening of the sigmoid loop , and externally applied abdominal pressure . in the available literature , only 6 patients were reported to have had some difficulty with intubation of the colon , while 21 specifically mentioned a lack of difficulty . reports from the remaining 10 cases made no mention of ease or difficulty of the procedure . twelve of the 37 reported patients had undergone previous surgery or had a disease process that may have enabled adhesion formation ( crohn 's disease and pancreatitis ) . eighteen had no predisposition to adhesion formation , and 8 reports lacked any information regarding risk factors . interestingly , of the 12 patients with possible adhesions , 10 had no intubation difficulties . most patients with colonoscopic splenic injury present relatively soon after injury with signs or symptoms suggestive of a problem . the range in the reported literature extends from within 2 hours to as long as 10 days . fourteen of the 32 patients with available information presented with symptoms within 12 hours of colonoscopy . the remaining 18 patients presented over the following days . the vast majority of patients with information available presented with symptoms of pain ( 28/32 ) . approximately half of these same patients also presented with evidence of hemorrhage or shock , or both ( 18/32 ) . with the exception of 1 patient successfully managed nonoperatively , the only reports that did not include pain as a presenting symptom were also those in which the patients died , suggesting the patients were too moribund to complain of pain . the diagnosis and management of these colonoscopy - related splenic injuries has to some degree reflected the available technology and is similar to the management of traumatic splenic injuries . although most injuries were diagnosed , or at least confirmed , at laparotomy in the era before 1987 , 21 of the 24 cases since 1989 have been diagnosed with noninvasive methods , such as computed tomography or ultrasonography . before the report of federle in 1983 , diagnosis of splenic injury sustained in trauma was indirect and was prompted by a positive diagnostic peritoneal lavage ( dpl ) leading to laparotomy . with the proven utility of ct in blunt abdominal trauma , only hemodynamically unstable patients now undergo dpl , and even that has been largely replaced by the focused abdominal sonography for trauma ( fast ) examination . the recognition over 100 years ago of the spleen 's role in immunoprotection against encapsulated organisms , such as streptococcus pneumoniae , haemophilus influenzae , and neisseria meningitidis was largely ignored until the 1950s when a swing in the management of splenic injuries began the era of splenic salvage . besides splenorrhaphy , splenic salvage maneuvers now include nonoperative management of splenic injuries and splenic artery embolization of pseudoaneurysms . in the trauma literature , nonoperative management of lower grade injuries ( grades i though none of the injuries induced at colonoscopy were graded in the available reports , these are presumably not the pulverized high - grade injuries seen in trauma , and probably fall into the grade i thru iii category . however , overall only 27.8% of patients with a splenic injury by colonoscopy have retained their spleens . before 1988 , that rate has dropped to 61.5% , still higher than that predicted from the trauma literature . finally , our patient presented with a condition not previously reported in the colonoscopy literature . the formation of a secondary cyst is rare and is characterized by the lack of a cellular lining as seen in a primary cyst . successful percutaneous drainage under ultrasound guidance has been reported for splenic cysts secondary to blunt abdominal trauma , but not for those related to colonoscopy . our attempt at percutaneous drainage was similarly intended to prevent splenectomy , but unfortunately was unsuccessful . with only limited experience , this technique , however , should remain in the armamentarium for the treatment of these injuries . in the history of splenic injury by colonoscopy , the experience of the first decade was laparotomy and splenectomy in all patients . though ct scanning has proven successful in diagnosing the injury , relatively few patients have escaped the experience with an intact spleen . nonoperative management and splenic artery embolization have been used with significant success in the trauma setting , but used only sparingly with colonoscopic injuries . though our attempt at percutaneous control of the secondary cyst formed as a result of a colonoscopic splenic injury was unsuccessful , we believe this could nevertheless represent an alternative to splenectomy in future patients .

the perspective presented here is that modern genetics is at a similar stage of development as were early formulations of quantum mechanics theory in the 1920s and that in 2010 we are at the dawn of a new revolution in genetics that promises to enrich and deepen our understanding of the gene and the genome . the interrelationships and interdependence of two views of the gene the molecular biological view and the epigenetic view are explored , and it is argued that the classical molecular biological view is incomplete without incorporation of the epigenetic perspective and that in a sense the molecular biological view has been evolving to include the epigenetic view . intriguingly , this evolution of the molecular view toward the broader and more inclusive epigenetic view of the gene has an intriguing , if not precise , parallel in the evolution of concepts of atomic physics from newtonian mechanics to quantum mechanics that are interesting to consider .

in a classic little book , what is life ? the great theoretical physicist erwin schrdinger considered one of biology 's central questions : how can a living organism be accounted for by physics and chemistry ? ( schrdinger , 1944 ) . in attempting to answer this question , schrdinger rightly focused on the centrality of the gene , and thus on the necessity of understanding the nature of mutations given that they are essential for identifying and investigating the nature of genes . because the chemical nature of the gene was unknown the paper by avery et al . ( 1944 ) demonstrating that dna was the genetic substance was published while what is life ? was still in press schrdinger drew on max delbrck 's atomic physics - based model of genetic mutation and the structure of the gene ( timofeeff - ressovsky et al . , 1935 ) suggesting that the gene is a linear one - dimensional crystal . i.e. , one whose elements do not repeat in a periodic way as in common crystals . although schrdinger 's little book was not universally well received , especially by biologists , even a critic like linus pauling was willing to conclude that schrdinger 's formulation of the theory of wave mechanics in 1925 , for which he received the nobel prize in 1933 , was basically responsible for modern biology because it developed largely on the basis of the new understanding of chemistry that quantum mechanics made possible ( pauling , 1987 ; cited in dronamraju , 1999 ) . quantum mechanics was certainly also at the core of schrdinger 's speculations on the nature of the gene . molecular biologists , many of whom had been trained as physical scientists , especially crystallographers and physical chemists ( such as max delbrck , linus pauling , francis crick , and matthew meselson , to name just a few ) . this was the dawn of the great molecular biology revolution that led to a vast increase in our understanding of genes and genomes and established the fundamental nature of the gene as a nucleic acid molecule comprised of a string of distinct nucleotide bases whose sequence normally specifies a gene product which can effect or influence the expression of phenotype during growth and development . interestingly , a complementary way of thinking about the nature of the gene that came to be associated with the term epigenetics also had its origins in the 1930s and 1940s . the epigenetic perspective pointed toward a more complicated reality for the nature of genes that was largely sidelined for several decades by the molecular biology revolution for good reason , because it was molecular biology that would have to develop the tools and approaches to eventually understand the molecular basis of the epigenetic perspective . thus , the two perspectives existed in parallel , with little cross - talk , simply because molecular biology had to develop and mature to the point that it could consider and address the epigenetic perspective . as first noted by brink ( 1960 ) , the epigenetic perspective was presaged by none other than morgan ( 1934 ) , when he noted the possibility that the genes also are building up more and more , or changing in some way as development proceeds , in response to that part of the protoplasm in which they come to lie . this view of the gene as dynamically changing in development without losing its fundamental properties ( morgan , 1934 ) has been undergoing a renaissance in recent years , as it has become increasingly clear that the dna - focused understanding of the gene that had revolutionized genetics and biology is actually incomplete without its complement , the chromatin-centered perspective of epigenetics . the chromatin - centered perspective on the gene was first elaborated 50 years ago by r. alexander brink . in a classic paper , brink ( 1960 ) inferred that eukaryotic chromosomes not only have a genetic function , but also possess what he termed a he proposed that a genetic locus was thus comprised of two types of chromatin , which he termed orthochromatin and parachromatin . brink defined orthochromatin as the substance at a locus that remains qualitatively constant in all nuclei of an individual and is presumably comprised of the dna at a given locus . parachromatin , on the other hand , brink proposed to be comprised of alternative states of chromatin that can be altered by factors arising in development or in response to the environment , and that these states can adopt a succession of mitotically transmissible states , together reflecting the progressive history of a cellular lineage . brink explicitly noted that the concepts of euchromatin and heterochromatin are fundamentally different than the concepts of orthochromatin and parachromatin , especially in that the locations of the former pair are mutually exclusive , whereas the latter pair are co - located intimately at the same genetic locus . brink based his hypothesis of a paragenetic function for chromosomes on observations that he and two other maize geneticists , barbara mcclintock and edward coe , made in the 1950s , which demonstrated the existence of two types of genetic variations , classical mutations and what he termed paramutations . paramutations , in the broadest use of the term ( brink , 1960 ) , are directed , for instance by particular paramutations are also typically reversible and changeable , often exhibiting a series of quantitatively or qualitatively varying states . brink argued that although paramutations violate mendel 's laws , their occurrence implies the existence of processes by which genes change in development and supports morgan 's contention that the genes might change in development without losing their fundamental ( i.e. , genetic ) properties . brink 's definition of parachromatin allowed great latitude in the nature , composition , and dynamic behaviors of parachromatin . in modern terms , we would include not only histone proteins and their various modification states , but also the many , diverse chromatin proteins and complexes that remodel chromatin into new states that may be comprised of conformational alterations , covalent modifications , and/or changes in molecular composition . explicitly included in such chromatin states would be not only proteins , but also rna molecules of various types , including but not limited to sirnas produced by rna interference - related chromatin complexes . thus , the modern view of chromatin at a given locus is one of a highly dynamic entity that fits well with brink 's concept of parachromatin . even though brink had no understanding of the molecular nature of chromatin , his hypothesis was flexible enough to fully encompass our modern understanding of chromatin . brink 's concept of parachromatin offered a broad general perspective on the nature , behaviors , and functions of genes in eukaryotes . but it was impossible in 1960 for brink to specify any molecular details or mechanisms that comprise parachromatin . only 1 year later , jacob and monod ( 1961 ) offered a simple , straightforward molecular biological model of the control of gene expression in prokaryotes that revolutionized thinking about how genes were expressed and regulated and that seemed to have the potential to account for the gene regulation in complex eukaryotes as well . parachromatin was largely forgotten for decades , as molecular biologists and geneticists explored the jacob monod model of gene regulation in great detail , first in prokaryotes and then in eukaryotes . eventually , however , it came to be recognized that simple models based on transcription factors binding to specific dna sequences were insufficient on their own to explain the control of gene regulation in complex eukaryotes . molecular tools and approaches were developed that could begin to open a window on the higher order complexities of eukaryotic gene regulation . chromatin was gradually accepted , first as a mediator or modulator of transcription complex formation , and then as an active , dynamic participant in the entire transcription process . when the concept of the histone code or the histone language was developed around 2000 , molecular biology was finally beginning to catch up to brink 's ( 1960 ) concept of parachromatin . of course , we are still very far from being able to say that we fully understand the molecular basis of parachromatin , but i have little doubt that we have begun to open the door to a vast realm of possibilities waiting to be explored and to be defined molecularly and mechanistically . the chromatin - centered perspective on the gene was first elaborated 50 years ago by r. alexander brink . in a classic paper , brink ( 1960 ) inferred that eukaryotic chromosomes not only have a genetic function , but also possess what he termed a he proposed that a genetic locus was thus comprised of two types of chromatin , which he termed orthochromatin and parachromatin . brink defined orthochromatin as the substance at a locus that remains qualitatively constant in all nuclei of an individual and is presumably comprised of the dna at a given locus . parachromatin , on the other hand , brink proposed to be comprised of alternative states of chromatin that can be altered by factors arising in development or in response to the environment , and that these states can adopt a succession of mitotically transmissible states , together reflecting the progressive history of a cellular lineage . brink explicitly noted that the concepts of euchromatin and heterochromatin are fundamentally different than the concepts of orthochromatin and parachromatin , especially in that the locations of the former pair are mutually exclusive , whereas the latter pair are co - located intimately at the same genetic locus . brink based his hypothesis of a paragenetic function for chromosomes on observations that he and two other maize geneticists , barbara mcclintock and edward coe , made in the 1950s , which demonstrated the existence of two types of genetic variations , classical mutations and what he termed paramutations . paramutations , in the broadest use of the term ( brink , 1960 ) , are directed , for instance by particular paramutations are also typically reversible and changeable , often exhibiting a series of quantitatively or qualitatively varying states . brink argued that although paramutations violate mendel 's laws , their occurrence implies the existence of processes by which genes change in development and supports morgan 's contention that the genes might change in development without losing their fundamental ( i.e. , genetic ) properties . brink 's definition of parachromatin allowed great latitude in the nature , composition , and dynamic behaviors of parachromatin . in modern terms , we would include not only histone proteins and their various modification states , but also the many , diverse chromatin proteins and complexes that remodel chromatin into new states that may be comprised of conformational alterations , covalent modifications , and/or changes in molecular composition . explicitly included in such chromatin states would be not only proteins , but also rna molecules of various types , including but not limited to sirnas produced by rna interference - related chromatin complexes . thus , the modern view of chromatin at a given locus is one of a highly dynamic entity that fits well with brink 's concept of parachromatin . even though brink had no understanding of the molecular nature of chromatin , his hypothesis was flexible enough to fully encompass our modern understanding of chromatin . brink 's concept of parachromatin offered a broad general perspective on the nature , behaviors , and functions of genes in eukaryotes . but it was impossible in 1960 for brink to specify any molecular details or mechanisms that comprise parachromatin . only 1 year later , jacob and monod ( 1961 ) offered a simple , straightforward molecular biological model of the control of gene expression in prokaryotes that revolutionized thinking about how genes were expressed and regulated and that seemed to have the potential to account for the gene regulation in complex eukaryotes as well . parachromatin was largely forgotten for decades , as molecular biologists and geneticists explored the jacob monod model of gene regulation in great detail , first in prokaryotes and then in eukaryotes . eventually , however , it came to be recognized that simple models based on transcription factors binding to specific dna sequences were insufficient on their own to explain the control of gene regulation in complex eukaryotes . molecular tools and approaches were developed that could begin to open a window on the higher order complexities of eukaryotic gene regulation . chromatin was gradually accepted , first as a mediator or modulator of transcription complex formation , and then as an active , dynamic participant in the entire transcription process . when the concept of the histone code or the histone language was developed around 2000 , molecular biology was finally beginning to catch up to brink 's ( 1960 ) concept of parachromatin . of course , we are still very far from being able to say that we fully understand the molecular basis of parachromatin , but i have little doubt that we have begun to open the door to a vast realm of possibilities waiting to be explored and to be defined molecularly and mechanistically . the evolution from mendelian genetics toward what we might call molecular epigenetics via molecular biology 's description of the gene as a nucleic acid molecule has an intriguing and perhaps instructive parallel in the evolution of physics from newtonian mechanics toward quantum mechanics via the planetary model of the atom in which electrons orbit the nucleus . the original concept of the atom , the fundamental entity of newtonian mechanics , had held that the atom was an indivisible particle . this concept was transformed twice first into the planetary model and then by quantum mechanics which redefined the atom as a field of probabilities of subatomic particles existing in four dimensions ( three spatial dimensions and time ) . similarly , the original concept of the gene , the fundamental entity of mendelian genetics , was that it was also particulate and indivisible , a concept that has also been transformed twice first through the prism of genetics and molecular biology into the concept of a gene as a nucleotide sequence , divisible by recombination , and now again by molecular biology , looking through the prism of epigenetics , into what might be referred to as a field of possibilities of alternative chromatin states centered on a particular nucleic acid sequence , i.e. , parachromatin , to use brink 's terminology . as mentioned previously , alternative parachromatin states are assumed to be based on diverse chromatin proteins , rnas , and complexes that can exist in various conformational and covalent modification states that are adopted during growth and development and can differ among loci . in quantum mechanics , subatomic particles may be viewed in two very different ways , i.e. , as both waves and particles ; likewise , paramutations may be viewed both as gene expression states and as mitotically , and in some cases , meiotically heritable states . classical genetics and then molecular biology established the macro - framework of genetics in the same way that newtonian mechanics and then the planetary model of the atom established the macro - framework of physics . quantum mechanics ultimately led to the idea of wave - particle duality and the description of positions of subatomic particles as a field of probabilities that determine the chemical properties of a given atom . similarly , brink introduced the idea of a genetic paragenetic duality of genetic loci in which a genetic entity ( dna ) and a paragenetic entity ( chromatin ) , both present at each genetic locus , are complementary and interdependent partners which we might describe as a field of possibilities that determine the expression states of a given locus . in drawing this parallel , i am not trying to suggest that the classical , molecular biological view of genetics is neither correct nor useful , any more than newtonian mechanics or the planetary model of the atom is neither correct nor useful . rather , i am simply suggesting that each represents early , incomplete descriptions of reality that required important modification and enrichment before we could fully understand the nature of the genetical and physical worlds , respectively . however , we ought not try to take the analogy too far because at some level of detail it most likely will break down . in the twentieth century , physics evolved beyond a deterministic view of the universe postulating that the future of the universe in theory could be extrapolated from the laws of physics if only one could obtain complete knowledge of the positions , directions , and velocities of movement of all particles in the universe . quantum mechanics , especially in heisenberg 's uncertainty principle raised fundamental questions that challenged the possibility of precise knowledge of the future : for instance , the number of times per second that atoms in a lump of uranium will undergo radioactive decay is known with precision ; however , why and when any particular atom will decay is unpredictable by modern physics . similarly , although geneticists can measure mutation frequency in a particular system under specific conditions , the timing of a particular nucleotide substitution ( or any other mutational event ) is unpredictable . thus , from an evolutionary genetic perspective , biology is no more deterministic than is physics , as tautz ( 2000 ) has analyzed in terms of population genetic theory . tautz argued that the selective fitness of an advantageous mutation in a population and time comprise a canonically conjugated pair of variables analogous to such pairs of physical variables , such as the location and momentum of a particle , or the energy of a particle and the time at which it was measured . he showed that uncertainty is largest at low allelic frequency and when the selective advantage is small . the specific trajectory of such an allele in a population of finite size is well known in population genetics to be unpredictable , and only probabilities for different trajectories can be determined . with the advent of genomics , it is theoretically possible to know with absolute certainty the sequence of a region of chromosome carrying a gene and even the sequence of an entire chromosome . however , as stadler ( 1954 ) noted , it is not trivial to precisely locate a gene , i.e. , it can not be shown to be delimited from neighboring genes by definite boundaries . this conclusion follows from stadler 's definition of the gene : operationally , the gene can be defined only as the smallest segment of the gene - string that can be shown to be consistently associated with the occurrence of a specific genetic effect . in modern terms , knowing the complete sequence of a chromosome does not allow us to precisely determine all of the many interdependent elements of a gene , including all those elements in cis that are necessary for the normal operation of a given gene that is associated with a specific genetic effect ( jorgensen , 2010 ) . in addition , the expression and selective value of a gene in nature may often be dependent on the environment encountered by the organism , perhaps making it impossible to precisely identify the boundaries of a gene . distinct from quantum mechanics , it is also important to recognize the relevance to biology of complexity theory , which has identified another type of uncertainty in physics , resulting from sensitive dependence on initial conditions such that relatively simple newtonian systems may exhibit unpredictable chaotic behaviors due to the impracticality of knowing initial conditions precisely enough . similarly , it should be evident that knowing all alternative epigenetic states of a given gene in all environments may be unachievable in any practical sense . this is essentially no different than what is postulated in complexity theory , namely , that it is impractical to know with enough precision the locations and movements of all particles of a system in order to predict future behavior with any certainty , at least in certain systems and under certain conditions . perhaps then , the most we can hope to achieve is to determine or estimate the field of possibilities that comprise the orthochromatin and parachromatin components of each gene . the marriage of epigenetics and molecular biology promises to deepen and revolutionize our understanding of the fundamental nature of the gene , allowing us to see deeply into its field of possibilities . it is going to be very interesting to learn what degree of knowledge of the field of possibilities that comprise a gene we will be able to obtain . though i will be happy to be proved wrong , my strong suspicion is that while we will be able to learn a lot about a given gene , we can never know what we do not know about it , i.e. , its complete field of possibilities will be not be determinable simply because it will be impossible to anticipate for every gene every circumstance that an organism may encounter . finally , given the rapidly increasing interest in the phenomenon of transgenerational ( meiotic ) inheritance of epigenetic and paragenetic states and their possible evolutionary consequences , it is also interesting to consider that epigenetics may also revolutionize our understanding of biological evolution ( perhaps similar to how understanding of atomic particles continues to revolutionize our understanding of the evolution of the universe ) . many biologists , especially plant biologists , have long postulated that information may sometimes flow backward from the environment to the gene via epigenetic impositions on the genome ( altering states of brink 's parachromatin ) and have suggested the possibility for the generation of novel genetic variations which , in the words of barbara mcclintock , might this seemingly radical suggestion ( which was not lamarckian , but rather a modified darwinian mechanism ) led to a great deal of criticism and misunderstanding of mcclintock unfair criticism , as i have argued previously ; rather than repeat that discussion here , i would refer the interested reader to jorgensen ( 2004 ) . the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

micrornas ( mirna ) are a novel class of small , noncoding rna molecules that have gained the attention of many researchers in recent years due to their ability to posttranscriptionally regulate the expression of families of genes simultaneously . their role in normal physiology and pathobiology is intriguing and their regulation in normal and disease states is fascinating . that the cells can return to a state of homeostasis when these small molecules are perturbed is truly remarkable given the multiple cellular targets of each mirna and that many mrnas are targeted by multiple mirnas . several reviews have covered aspects of mirna function in biology and disease . here , we review the role of mirna in regulating the renin - angiotensin system , age / rage signalling , and under conditions of oxidative stress in the context of diabetic nephropathy .

the world health organization states that ~347 million people , roughly 9.5% of the adult population , were suffering from diabetes in 2008 . the incidence of diabetes is rapidly increasing with estimates suggesting that this number will almost double by 2030 . diabetes mellitus is a major cause of chronic kidney disease ( ckd ) worldwide and is associated with enhanced morbidity and mortality , in particular accelerated cardiovascular disease [ 2 , 3 ] . diabetic nephropathy ( dn ) is now the most common cause of end - stage renal failure in the western world . clinical associations that frequently precede overt dn are hypertension and poor glycaemic control , although a subset of patients develop nephropathy despite the proper glycemic control and normal blood pressure . once nephropathy is established , blood pressure often rises further , but glycaemic control can paradoxically improve as a result of reduced renal insulin clearance . it is postulated that the interplay between metabolic and hemodynamic pathways plays an important role in the development and progression of dn ( figure 1 ) . increased systemic and intraglomerular pressure activation of the renin - angiotensin - aldosterone system ( raas ) has been recognized as a key component of dn progression . additionally , chronic hyperglycemia promotes the generation of advanced glycation end - products ( ages ) . it is widely accepted that ages mediate their effects both directly and indirectly through receptor - dependent mechanisms . the receptor for age ( rage ) acts as a signal transduction receptor , and the rage - age interaction activates multiple intracellular signalling pathways which increase the production of growth factors , inflammatory cytokines , and oxidative stress ( figure 1 ) . in recent times , a novel class of non - coding rna , microrna ( mirna ; mir ) , has been found to be expressed in all tissues and plays important roles in tissue homeostasis and disease progression [ 9 , 10 ] . whilst the role of mirna in the pathogenesis of dn has been extensively reviewed by others in relation to growth factors and fibrosis in dn , the focus of this review is on the role of mirna in the renin - angiotensin system and the age / rage signalling pathway , and their downstream the mediators such as oxidative stress and the immune response in the context of diabetic nephropathy . mirnas are a group of small ( ~22 nucleotide ) single - stranded non - coding rnas ubiquitously expressed in plants and animals where they act posttranscriptionally to modulate the expression of target genes [ 11 , 12 ] . they were first discovered in 1993 when lin-14 protein expression was found to be regulated by the mature product of the lin-4 gene in caenorhabditis elegans ( c. elegans ) . the mechanism was found to rely upon sequence specificity in the 3-untranslated region ( utr ) of lin-14 to which lin-4 bound . whilst this occurred with only partial complementarity , the binding occurred with sufficient affinity to result in inhibition of protein translation . at the time this was considered a peculiarity in the worm . it was not until 2000 when another such regulator , let-7 , was discovered in c. elegans , and soon after came the realisation that this form of regulation was conserved across many species , representing a general mechanism for regulating the expression of several genes . mirnas are found in intergenic sequences or on the antisense strand of genes and may possess their own promoter and regulatory sequences [ 15 , 16 ] . other mirnas ( almost 50% in the case of human mirna ) are found within gene sequences and are together regulated with their host gene [ 1719 ] . approximately half are found in polycistronic units from which the mature mirnas are processed . the primary transcript ( pri - mirna ) is processed by a number of proteins including ribonuclease iii , drosha , and the rna binding protein , digeorge syndrome critical region gene 8 ( dgcr-8 ) protein , into a short hairpin rna molecule termed the precursor mirna ( pre - mirna ) which is subsequently exported from the nucleus by exportin-5 [ 2022 ] . once in the cytosol , further processing is mediated by another ribonuclease iii , dicer , and this is followed by the incorporation of the mature strand of the duplex mirna into the rna - induced silencing complex ( risc ) which includes the argonaute family of proteins . the mirna - risc complex stabilises the mirna against nuclease attack and mirna direct the complex to target rna transcripts via sequence complimentarity between the mirna seed sequence ( 28 nucleotides ) and the mirna recognition element ( mre ) in the target transcript . mirnas modulate protein synthesis by binding to the 3 utr of mrnas via incomplete base pairing to the complementary seed sequence in the utr , leading to translational repression . mres are predominately located in the 3 utrs of mammalian mrna , however ; there is evidence indicating that mirna can also mediate translational repression via binding 5 utrs or coding sequences [ 25 , 26 ] . in plants and lower vertebrates where complete complimentarity exists between an mirna seed sequence and the target mrna , degradation of the target mrna in contrast , mirnas exercise finer control on protein expression in mammalian cells where they repress protein translation [ 27 , 28 ] , allowing for more comprehensive regulation of protein expression as any single mirna can target many mrnas , and any mrna can be the target of several mirnas . this potential of mirnas to regulate many genes adds significant complexity to the interpretation of many studies where the focus is on single genes . it is therefore critical to computationally detect the combination of mirnas that target specific mrna molecules . such analyses often demand complex algorithms that need to be defined by high stringency levels to generate computational data that could then be assessed and validated in the wet lab . added to this is the recent discovery of circulating mirnas in microparticles and the potential of these mirnas to be delivered to sites distal to their generation . renin production is the key regulatory step that initiates an enzymatic cascade that leads to angiotensin generation and the control of blood pressure in addition to fluid and electrolyte homeostasis . renin is synthesized and released by renal juxtaglomerular ( jg ) cells , which are located at the entrance to the glomerulus . in early embryonic development , renin producing cells are broadly distributed along intrarenal arteries in the glomerular mesangium and in a few developing tubules . with maturation lineage studies demonstrate that this progressive restriction is achieved by differentiation of renin cells into vascular smooth muscle cells ( vsmcs ) , mesangial cells , and a subset of tubular cells . were the first to address the role of mirna in the maintenance of jg cells by generating mice with a conditional deletion of dicer ( dicer cko mice ) specifically and exclusively in renin - expressing cells , resulting in selective ablation of mirna generation only in renin cells and their descendants . deletion of dicer resulted in severe reduction in the number of jg cells accompanied by decreased expression of the renin ren1 and ren2 genes , decreased plasma renin concentration ( prc ) , decreased bp , abnormal renal function , and striking nephrovascular abnormalities including striped corticomedullary fibrosis . this study clearly showed a critical role of mirna in the orchestration of renal function and the importance of mirna homeostasis for specific organ functions . marques and colleagues conducted for the first time transcriptome - wide study of differential expression of mrnas and mirna in kidneys of hypertensive subjects ( 15 untreated hypertensive and 7 normotensive white males ) by microarray technology . they confirmed differences of expression for nuclear receptor subfamily 4 group a member 1 ( nr4a1 ) , nr4a2 , nr4a3 , period circadian protein homolog 1 ( per1 ) , and salt - inducible kinase 1 ( sik1 ) mrnas and for the mirnas hsa - mir-638 and hsa - let-7c by real - time quantitative pcr expression in the medulla . functional experiments confirmed the predicted binding of hsa - let-7c to the 3 untranslated region of nr4a2 mrna . in the renal cortex they confirmed differences in expression of apoptosis - inducing factor , mitochondrion - associated , 1 ( aifm1 ) , alpha-1-microglobulin / bikunin precursor ( ambp ) , apolipoprotein e ( apoe ) , cluster of differentiation 36 ( cd36 ) , ephrin - b1 ( efnb1 ) , nadh dehydrogenase ( ubiquinone ) complex i , assembly factor 1 ( ndufaf1 ) , peroxiredoxin 5 ( prdx5 ) , ren , renin binding protein ( renbp ) , solute carrier family 13 ( sodium / sulfate symporters ) , member 1 ( slc13a1 ) , syntaxin 4 ( stx4 ) , and troponin t type 2 ( cardiac ) ( tnnt2 ) mrnas , and the mirnas hsa - mir-21 , hsa - mir-126 , hsa - mir-181a , hsa - mir-196a , hsa - mir-451 , hsa - mir-638 , and hsa - mir-663 . functional experiments demonstrated that hsa - mir-663 can bind to the ren and apoe 3 untranslated regions regulating ren and apoe mrna levels , whereas hsa - mir-181a regulated ren and aifm1 mrna . a major discovery was evidence for ren mrna regulation via binding of mirnas hsa - mir-181a and hsa - mir-663 to its 3 utr as the observed downregulation of these 2 mirnas in hypertension could explain the elevation in intrarenal renin mrna . due to small sampling size , these findings need to be bolstered by the acquisition of suitable samples from larger cohorts of untreated hypertensive subjects in other settings . chronic kidney disease ( ckd ) is a known cardiovascular risk factor , and most patients with ckd die of cardiovascular disease ( cvd ) before reaching the need for dialysis . chen and colleagues measured vascular mirnas in blood from 90 patients with ckd and found an association between decreased circulating levels and progressive loss of egfr by multivariate analyses . expression of vascular mirnas was decreased in the thoracic aorta of ckd rats compared to normal rats , with concordant changes in target genes of runx2 , at1r , and myocardin with no alteration in drosha or dicer , indicating that the low levels of expression are not due to altered intracellular processing . furthermore , the expression of mir-155 was negatively correlated with calcification of the aorta , a process known to be preceded by vascular dedifferentiation in these animals . overexpression of mir-155 in vsmc from ckd rats inhibited at1r expression and decreased cellular proliferation , confirming a causative role of low mir155 in vsmc transformation to a more synthetic , proliferative phenotype . however , whether downregulation of these mirnas is the cause or consequence of the widespread vascular phenotype abnormalities in patients with ckd remains to be determined . jeppesen and colleagues have shown that angii regulates five mirnas ( mir-29b , -129 - 3p , -132 , -132 - 5p , and -212 ) during in vitro stimulation of primary cardiac fibroblasts and of hek293n cells overexpressing the at1r . furthermore , eskildsen and colleagues undertook a detailed analysis of potential mirnas involved in angii - mediated hypertension in rats and hypertensive patients , using mirna microarray and qpcr analysis . mir-132 and mir-212 are highly increased in the heart , aortic wall , and kidneys of rats with hypertension ( 159 12 mm hg ) and cardiac hypertrophy following chronic angii infusion . in addition , activation of the endothelin receptor , another gq coupled receptor , also increased mir-132 and mir-212 . a significant downregulation of mir-132 as well as a robust attenuation of mir-212 in human arteries from the arb - treated patients was also observed , whereas treatment with -blockers had no effect . in conclusion , mir-132 and mir-212 are upregulated in angii - induced hypertension in organs associated with blood pressure control , possibly via the gq - dependent pathway . table 1 contains mirnas that are known to be regulated by the renin - angiotensin system in the kidney and other tissues and associated with kidney disease , hypertension , and cardiovascular disease . chronic hyperglycemia promotes the generation of advanced glycation end products ( ages ) as a result of sequential biochemical reactions involving nonenzymatic glycation of protein and lipids known as the maillard reaction . as a consequence of age formation ages can induce expression of the mcp-1 in podocytes through activation of the age receptor ( rage ) and generation of intracellular reactive oxygen species ( ros ) . the induction of oxidative stress results in upregulation of nuclear factor ( nf)-b and various nf-b - mediated proinflammatory genes , eventually leading to glomerular and tubulointerstitial injury . therefore , age / rage and oxidative stress signalling are important in the progression of dn and targeting this axis by modulating the mirnas that are involved is a potential new therapy . there are very few studies examining the role of mirna in age / rage signalling related to kidney disease . most studies have focussed on the role of rage in the immune system or cancer because rage is a member of the immunoglobulin superfamily of cell surface molecules . when dicer was selectively inactivated in mouse podocytes , multiple abnormalities were observed in glomeruli of mutant mice , including foot process effacement , irregular and split areas of the glomerular basement membrane , podocyte apoptosis and depletion , mesangial expansion , capillary dilation , and glomerulosclerosis . gene profiling by microarray analyses revealed upregulation of 190 genes in glomeruli isolated from mutant mice at the onset of proteinuria compared with control littermates . target sequences for 16 mirnas were significantly enriched in the 3-untranslated regions of the 190 upregulated genes . further , supporting the validity of the in silico analysis , 6 of the 8 top - candidate mirnas were identified in mirna libraries generated from podocyte cultures ; these included mir-28 , mir-34a , and four members of the mir-30 family , mir-30c-1 , mir-30b , mir-30d , and mir-30c-2 . among the 15 upregulated target genes of the mir-30 mirna family , rage , vimentin , heat - shock protein 20 , and immediate early response 3 were known to be expressed in injured podocytes in experimental models and human kidney disease . rage and immediate early response 3 are known to mediate podocyte apoptosis , whereas vimentin and heat - shock protein 20 are involved in cytoskeletal structure . the findings demonstrate the important roles of the mir-30 family in podocyte homeostasis and podocytopathies . s100b , a rage ligand , significantly increased cox-2 mrna accumulation in thp-1 monocytes at 2 h via mrna stability . s100b decreased occupancy of the dna / rna - binding protein , heterogeneous nuclear ribonuclear protein k ( hnrnpk ) , at the cox-2 promoter but simultaneously increased its binding to the cox-2 3-utr . additionally , s100b significantly downregulated the expression mir-16 which acts to destabilize cox-2 mrna by binding to its 3-utr . these results demonstrate that diabetic stimuli can efficiently stabilize inflammatory genes via opposing actions of key rna - binding proteins and mirna . ages delay spontaneous apoptosis of monocytes and contribute to the development of inflammatory responses . in genome - wide mirna expression analysis significant upregulation of mir-214 was consistently observed in thp-1 and human monocytes treated with various ages . a striking increase in mir-214 overexpression of pre - mir-214 led to impaired pten expression and delayed apoptosis of thp-1 cells , whereas knockdown of mir-214 levels largely abolished age - induced cell survival . these findings define a role for mir-214-targeting of pten in age - induced monocyte survival . high - mobility group box 1 protein ( hmgb1 ) is a late inflammatory cytokine that signals danger to the immune system through the rage and toll - like receptor . mir-221 and mir-222 are involved in cell proliferation through the inhibition of the cell cycle regulator , p27kip1 , in smooth muscle cells and endothelial cells ( ecs ) . hmgb1 increases the expression of mir-221 and mir-222 in primary cultures of excised papillary lesions and in an established papillary cancer cell line and overexpression of mir-222 and mir-221 caused by hmgb1 increases growth and motility in papillary thyroid cancer cells . recently , it was reported that mir-21 and mir-221 , which are tissue inhibitors of metalloproteinases ( timp)3-targeting mirna , were significantly upregulated in kidneys from diabetic mice compared to control littermates and in a mesangial cell line grown in high glucose conditions . mesangial expansion is one of the main characters in dn and is mainly due to accumulation of extracellular matrix ( ecm ) . ecm turnover is regulated by timps activities . in diabetic conditions , timp3 expression in kidney mir-221/mir-222 cluster which has been examined in several cardiovascular disorders affects the angiogenic activity of stem cell factor ( scf ) by targeting the receptor c - kit . high glucose levels elevate mir-221 and this correlates with decreased expression of c - kit and reduced ec migration . mir-221/222 targets no synthase and transcription factor ets-1 which are both major contributors to the atherosclerotic process . [ 47 , 49 , 50 ] taken together , these results indicate that hmgb1/rage and mir-221/222 axis may be activated in the kidney and vasculature resulting in ecm accumulation and atherosclerosis in dn patients . table 2 lists the mirnas that are known to be modulated by the age / rage in kidney , monocytes , vsmcs , and ecs and associated with kidney disease and vascular dysfunction . reactive oxygen species ( ros ) is a collective term that includes a number of reactive and partially reduced oxygen ( o2 ) metabolites . some of them are free radicals , such as superoxide anion ( o2 ) and hydroxyl radicals ( oh ) that are extremely reactive molecular species with an unpaired electron in their outer orbital . a number of studies have demonstrated that both type 1 and type 2 diabetes are associated with overproduction of oxygen - derived free radicals . increased ros production and reduced levels of antioxidants culminate with an in increased level of oxidative stress leading to oxidative damage to cellular components . among the many enzymatic systems implicated in ros generation in vascular tissues , enzymes of the mitochondrial respiratory chain ( complexes i and iii ) , xanthine oxidase , uncoupled nitric oxide synthase , and nicotinamide adenine dinucleotide phosphate reduced form ( nadph ) oxidase ( nox ) appear to be particularly important [ 5254 ] . increased nox - mediated superoxide production has been reported in experimental models of diabetes and occurs in parallel with upregulation of nox1 and nox4 [ 55 , 56 ] . nox - mediated generation of superoxide is an important mediator of matrix accumulation , renal fibrosis , and podocyte injury in dn . mir-377 is upregulated in spontaneous and stz - induced mouse models of dn and in mesangial cells exposed to high glucose and tgf-1 . furthermore , genes potentially relevant to the pathogenesis of dn were confirmed experimentally , including the cytoskeletal regulator p21-activated kinase 1 ( pak1 ) and superoxide dismutases ( sod1/sod2 ) that catalyse ros , which accumulates in response to hyperglycemia . it has previously been shown that upregulation of the mir-23a~27a~24 - 2 cluster induces caspase - dependent and caspase - independent cell death in human embryonic kidney cells via c - jun n - terminal kinase ( jnk ) with increases in ros and the release of proapoptotic factors such as cytochrome c ( cyt c ) in addition to apoptosis - inducing factor ( aif ) from the intermembrane space of mitochondria to the cytosol . in order to better understand the molecular mechanism responsible for mir-23a~27a~24 - 2 cluster - induced cell death , gene expression profiling was performed in control and mir-23a~27a~24 - 2 cluster overexpressing hek293 t cells . this revealed mir-23a~27a~24 - 2 cluster - induced apoptosis was associated with endoplasmic reticulum ( er ) stress and the unfolded protein response ( upr ) pathways in hek293 t cells . overexpression of the mir-23a~27a~24 - 2 cluster resulted in er stress and altered mitochondrial membrane permeability and this was further established by increased intracellular and mitochondrial calcium levels in hek293 t cells . there have been reports that mir-23b levels were increased in the kidneys of tgf-1 transgenic mice and rats following subtotal nephrectomy , which was found to be localized to podocytes and tubular epithelium by in situ hybridization . mir-23b was also upregulated by tgf-1 in cultured renal epithelial cells . in vitro gain and loss - of - function studies pointed to several mir-23b targets , including tgf- receptor type ii , smad3 , and tgf-1 itself , suggesting a negative feedback loop - regulating tgf-1 signaling . modulation of mir-23b in cultured podocytes altered expression of wt1 , nephrin , and podocin and also influenced motility of cultured tubular cells . studies have found that in the aged organs , including the kidney , expression of antioxidant enzymes such as sod , catalase , gpx , and peroxiredoxins is downregulated [ 62 , 63 ] thus leading to reduced antioxidant capacity . from bioinformatic analysis of the mirna expression profile of young and old rat kidneys , mitochondrial sod2 and thioredoxin reductase 2 ( txnrd2 ) are potential targets of mir-335 and mir-34a , respectively , as aging mesangial cells exhibited significant upregulation of mir-335 and mir-34a and marked downregulation of sod2 and txnrd2 . further studies confirmed sod2 and txnrd2 as target genes of mir-335 and mir-34a which coincided with ros generation . within the mitochondria , uncoupling protein 2 ( ucp2 ) has recently been reported as a negative regulator of ros generation . its ablation leads to marked increase of oxidative stress in several cell types . in the stroke - prone spontaneously hypertensive rat ( shrsp ) kidneys , severe renal damage along with increased rate of inflammation and oxidative stress ucp2 gene and protein levels were downregulated paralleled by differential expression of kidney mir-24 and -34a , which were identified to target the ucp2 gene . the silencing of the ucp2 gene in renal mesangial cells led to increased rate of ros generation , increased inflammation and apoptosis , reduced cell vitality , and increased necrosis , suggesting that ucp2 is critical in preventing oxidative stress damage in renal mesangial cells . specific mirnas , including mir-205 and the mir-200 family ( mir-200a c , mir-141 , and mir-429 ) , were shown to mediate epithelial - to - mesenchymal transition ( emt ) in response to tgf-1 in madin - darby canine kidney cells . downregulation of these mirnas relieves their cooperative repression of the mesenchymal transcription factors zeb1 and sip1 that , in turn , are free to inhibit e - cadherin expression promoting epithelial dedifferentiation . assessed changes in mirna expression in the cultured renal tubular cell line hk-2 under hypoxia - reoxygenation - induced oxidative stress or er stress using mirna microarray assay and real - time rt - pcr . among altered mirna expression , mir-205 was markedly decreased in both stress conditions . functional analysis revealed that decreased mir-205 led to an increase in cell susceptibility to oxidative and er stresses and that this increase was associated with the induction of intracellular ros and suppression of antioxidant enzymes . furthermore , mir-205 bound to the 3-utr of the prolyl hydroxylase 1 ( phd1/egln2 ) gene and suppressed the transcription level of egln2 , which modulates both intracellular ros level and er stress state . mirna profiling of huvec treated for 8 and 24 hrs with 200 m hydrogen peroxide ( h2o2 ) showed that mir-200c and the cotranscribed mir-141 increased more than eightfold . mir-200c overexpression in huvec recapitulates many aspects of the oxidative stress - induced phenotype , since it induces cell growth arrest , apoptosis , and cellular senescence . all these effects are mediated , at least in part , by the inhibition of the target zeb1 by the mir-200 family . mir-200 family induction following h2o2 exposure has been confirmed in different cell lines such as human and mouse immortalized fibroblasts , colon carcinoma ( ct26 ) , mammary gland epithelial cells ( nmumg ) and human cell lines , melanoma cells ( mda - mb-435s ) , kidney cells ( 293 t ) , breast adenocarcinoma ( mda - mb-436 and bt-549 ) , and ovarian adenocarcinoma ( skov3 ) . notably , in all these cell lines , all mir-200 family members were upregulated . in a recent study , an analysis of mirnas upregulated in diabetic mouse heart compared to control was performed revealing that mir-200c and mir-141 were among the most upregulated . the authors show that mir-141 targets the inner mitochondrial membrane phosphate transporter , solute carrier family 25 member 3 ( slc25a3 ) , which provides inorganic phosphate to the mitochondrial matrix and is essential for atp production , suggesting an important role of mir-200 family in mitochondrial responses involved in cardiac diseases associated with diabetes and obesity . on the contrary to the role for mir-200 family in oxidative stress , the mir-200 family also plays an important role in emt , which is considered to mediate production of renal fibroblasts , in part by targeting zeb1/2 , the transcriptional repressors of e - cad [ 68 , 77 , 78 ] . on the other hand , another group has demonstrated that tgf- activated akt in glomerular mesangial cells by inducing mir-200b and mir-200c , both of which target fog2 , an inhibitor of phosphatidylinositol 3-kinase activation , suggesting the role of mir-200 family on glomerular mesangial hypertrophy in the progression of dn . some of the differences may relate to variances in experimental models and/or conditions ; however , one often overlooked explanation is that some effects of mirna and inhibitors are likely to be indirect in nature . recent evidence demonstrates regulation of gene expression via deadenylation , by altering message stability , and by effects on transcription . bioinformatics utilising pathway analysis will be needed to better understand the crosstalk between factors that drive many downstream processes and how those processes ultimately impact the expression of individual genes . mirna profiling of rat vsmcs treated with 200 m h2o2 for 6 hours revealed an upregulation of mir-21 . this study showed that mir-21 participates in h2o2-mediated gene regulation via its target programmed cell death 4 ( pdcd4 ) and transcription factor ap-1 pathway activity . elevated mir-21 is thought to contribute to atherosclerosis by directly targeting ppar , leading to an increased inflammatory response in ecs . inhibition of mir-21 causes activation of ap-1 as well as upregulation of proinflammatory factors such as vcam-1 and mcp-1 . mir-21 can also act as an inhibitor of angiogenesis by reducing ec proliferation , migration , and tube formation in culture via inhibition of rhob . mir-21 can also increase nitric oxide ( no ) in huvecs exposed to shear stress via increased phosphorylation of nitric oxide synthase ( nos ) and decreased apoptosis . this activity is balanced by the ability of mir-21 to repress sod-2 which is important in antioxidant defence . furthermore , mir-21 is elevated in angiogenic precursor cells by asymmetrical dimethylarginine ( adma ) which is a powerful nos inhibitor , ultimately resulting in elevated intracellular reactive oxygen ( ros ) species . knockdown of mir-21 decreased mesangial expansion , collagen i / iv , and fn1 expression and reduced macrophage infiltration and tnf and mcp-1 expression . the gene expression changes were replicated in vitro in both ptc and mesangial cells ( mcs ) with mir-21 overexpression enhancing fibrogenesis via a mechanism which in part involved the direct targeting of smad7 . interestingly , mir-21 targets pten and also leads to decreased mesangial expansion in db / db mice . mir-21 has also been implicated in regulation of tgf- signalling in a number of animal models of tubulointerstitial fibrosis and associated renal dysfunction . in one such model , smad7 overexpression in the rat unilateral ureteral obstruction model has restored mir-21 expression to normal levels with congruent improvements in renal pathology . in line with a profibrotic role for mir-21 , upregulation of this mirna furthermore , regulation of pdcd4 by mir-21 enhances podocyte apoptosis and loss in conjunction with increased tubular epithelial cells survival against growth arrest signals [ 85 , 86 ] . mir-210 appears to function as master regulator of the hypoxic response as it was found to be upregulated by hypoxia in virtually all the cell types tested to date [ 87 , 88 ] . recent data demonstrate that hif1 can block both mitochondrial respiration via the electron transport chain ( etc ) through transcriptional activation of mir-210 in some cell types [ 87 , 88 ] . upregulation of mir-210 along with vegf and vegfr2 expression was confirmed in renal ischemia / reperfusion ( i / r ) injury of male balb / c mice . furthermore , overexpression of mir-210 in huvec-12 cells enhanced vegf and vegfr2 expression and promoted angiogenesis in matrigel in vitro . these results suggest that mir-210 may be involved in targeting the vegf signaling pathway to regulate angiogenesis after renal i / r injury . table 3 summarises the mirnas that are regulated by oxidative stress in the kidney and other tissues and associated with kidney disease and vascular dysfunction resulting from excessive ros production . considerable progress has been made in identifying a number of important roles for mirna in various biological processes and in disease . there is much excitement at the prospect that some mirnas appear to be important to the regulation of several related processes in diabetes and its complications , including the modulation of the raas and oxidative stress pathways . the number of mirnas relevant to these conditions is constantly increasing ( tables 13 ) . it is encouraging that in some cases restoring the expression of a dysregulated mirna can attenuate or even reverse disease . our initial understanding of gene regulation has continued to change from simple concepts in terms of protein factors sitting on dna to complex epigenetics involving chromatin dynamics and multiple histone and dna modifications . even this complexity has been superseded by the ability of mirna to modulate the expression of multiple targets posttranscriptionally . whilst the biology around mirna continues to generate new and interesting findings , the challenge of the future is to translate some of the exciting experimental findings to the potential therapeutic interventions .

phylogenomic approaches have shown that eukaryotes acquire genes via gene transfer . however , there are two fundamental problems for most of these analyses ; only transfers from prokaryotes are analyzed and the screening procedures applied assume that gene transfer is rare for eukaryotes . directed studies of the impact of gene transfer on diverse eukaryotic lineages produce a much more complex picture . many gene families are affected by multiple transfer events from prokaryotes to eukaryotes , and transfers between eukaryotic lineages are routinely detected . this suggests that the assumptions applied in traditional phylogenomic approaches are too nave and result in many false negatives . this issue was recently addressed by identifying and analyzing the evolutionary history of 49 patchily distributed proteins shared between dictyostelium and bacteria . the vast majority of these gene families showed strong indications of gene transfers , both between and within the three domains of life . however , only one of these was previously reported as a gene transfer candidate using a traditional phylogenomic approach . this clearly illustrates that more realistic assumptions are urgently needed in genome - wide studies of eukaryotic gene transfer .

these are two distantly related groups of eukaryotes that have similar lifestyles . both feed by osmotrophy . the cells secret enzymes that decompose organic matter and the metabolites are imported into the cell . fungi are more closely related to animals than to oomycetes , whereas diatoms , a group of photosynthetic algae , are a sister group to oomycetes . , the oomycete and fungi genomes are not among the genomes for which traditional phylogenomic studies have indicated a significant role of gene transfer in eukaryotes . nevertheless , targeted evolutionary studies have suggested that gene transfer contributed to the similarities between the groups . they could identify dozens of gene transfers between the groups using a wide range of genomes from both groups together with clustering and phylogenetic methods . interestingly , all transfers except one were reported to have occurred in the direction from fungi to oomycetes . many of the transferred genes encode secreted decomposing enzymes and were specifically acquired by plant - tissue colonizing oomycetes . these results show that oomycete most likely are more recent plant pathogens than fungi and that transfer of genetic material from a distantly related eukaryotic group have played an important role in evolution of their pathogenic lifestyle . these fascinating results would not have been obtained with a traditional phylogenomic approach in which genes with strong sequence similarities to other eukaryotes would have been assumed to be present in the common eukaryotic ancestor . studies of gene transfer are indeed able to shed light on the diversification process of eukaryotes . choanoflagellates are a group of free - living microbial eukaryotes which are the closest relatives to animals . sun and coworkers used a directed phylogenomic approach to search for genes of algal origin in the genome of monosiga brevicollis , a phagotrophic unicellular choanoflagellate . using realistic filtering criteria they were able to identify 103 genes with strong support for algal origin , mostly from haptophytes , diatoms and green algae . this could be the result of repeated transfer of genes from food ; choanoflagellates feed on bacteria and other eukaryotes . alternatively , or rather in addition , the genes could have been introduced from a past algal endosymbiont in the lineage leading to monosiga . interestingly , a quarter of the identified genes appeared to first have been transferred from bacteria to a eukaryotic alga , and then secondarily to choanoflagellates . however , such a bacterial origin would not have been detected using a typical phylogenomic approach since the strongest sequence similarity would be to algal gene of bacterial origin . functions in amino acid and carbohydrate metabolism dominated among the gene transfer candidates , indicating that these choanoflagellates have adapted by acquisition of algal genes that expand their metabolic repertoire . the two examples outline above test well - defined hypotheses about gene transfers by using directed phylogenomic methods in combination with careful filtering and interpretation of the results . they are very powerful to characterize the role of gene transfers between distantly related eukaryotic groups in the adaptation of eukaryotes . however , one disadvantage is that the approach relies on existing knowledge of the biology of the organisms ; if only transfers between two organismal groups are addressed important contributions from other groups may be missed in such phylogenomic approaches . in addition , these kinds of studies can only estimate minimal number of transfers between the groups analyzed ; the number of false negatives may be large . to circumvent these problems i applied an alternative approach to study these issues . i first identified patchily distributed proteins because these are expected to be enriched with gene transfer events ( fig . 1 ) , instead of screening for unexpected sequence similarities then i performed phylogenetic analyses for each identified gene family to evaluate whether the patchy distribution was a consequence of gene transfer , or differential loss in the eukaryotic domain . the soil - dwelling cellular slime mold d. discoideum was selected in the case study for two reasons : an active research community have produced a high quality annotation of the genome sequence ( http://dictybase.org/ ) , and only 18 potential gene transfers were reported in the original publication . i identified 49 protein families in the dictyostelium genome which were shared with at least one prokaryotic species , but only a limited number of other eukaryotes and prokaryotes ( fig . 1 ) . the evolutionary history of these patchily distributed families were analyzed further . for seven of the families the remaining 42 families contained sequences from one or more eukaryotic species outside the dictyostelium genus . the closest relative with a completely sequenced genome , the human parasite e. histolytica , was represented in only two families . in contrast , the amoeboflagellate naegleria gruberi had a representative in 25 of the families . dictyostelium and naegleria are having somewhat overlapping lifestyles , they are both free - living heterotrophs that can be found in soil and they both undergo cell differentiation under certain conditions . however , they are distantly related eukaryotes classified within two different supergroups : amoebozoa and excavata . there exist at least two alternative plausible explanations for this striking gene - sharing pattern . these genes were present in the common ancestor of the dictyostelium and naegleria and distributed in eukaryotes strictly by vertical inheritance . in lineages that have different lifestyles ( i.e. , parasites ) alternatively , the genes have been distributed via gene transfer in more recent evolutionary timescales providing selective advantage to the recipient lineages . the vast majority of the phylogenetic trees showed strong indications of lateral gene transfer between prokaryotes and eukaryotes and within eukaryotes.figure 1b shows an example of an individual gene tree . the exact details of the transfer events could in many cases not be traced , because the density of organismal sampling was too low . nevertheless , there are no strong indications that any of the proteins have evolved solely via vertical inheritance and gene loss ; gene transfer has likely affected all patchily distributed genes families identified in the analysis to some extent . only a single protein among the 49 identified as patchily distributed was among the 18 gene transfer candidates in the original d. discoideum genome publication , and very few were among the 184 lateral gene transfer candidates reported from n. gruberi . this may be surprising , but is logical if the details of the methods applied are considered . dictyostelium genes with significant similarity to a bacterial - specific pfam domain and only present in dictyostelium among eukaryotes were considered as gene transfer candidates . this conservative approach is unlikely to pick up false positives , but will be very prone to false negatives . genes acquired via gene transfer in two or more different eukaryotes are excluded , as are any genes without sufficient sampling among prokaryotes to be included in pfam . similarly , the n. gruberi gene set was screened with similarity searches , and genes with significant similarity only to prokaryotes were considered as gene transfer candidates . again , gene families with repeated transfers are missed in the screen and eukaryote - to - eukaryote transfers are not even considered . the true number of gene families in these microbial eukaryotes are likely much larger than has been reported . the spread of patchily distributed genes are part of this adaptation process , and there is no reason to assume that microbial eukaryotes do not take part of this flux of genetic material ( fig . 1 ) . for example , if a gene provide the ability to utilize a carbon compound present in the environment it is likely to spread to different microbes in the environment previously lacking this ability ( provided that there are mechanisms in action ) . the assumption that a gene has a vertical eukaryotic history is violated as soon as two eukaryotic lineages inhabit a similar environment and acquire their copy of a particular gene family independently during the adaptation process . the traditional phylogenomic approaches will fail to identify members of such protein families as gene transfer candidates because they assume that vertical inheritance is the norm for all protein families with gene transfer events as very rare exceptions . however , this is probably only the case for universal core genes , and certainly not for patchily distributed proteins ( fig . 1 ) . traditional phylogenomic approaches probably only have scratched the surface of the gene transfer events and thereby drastically underestimated the impact of the process on eukaryotic genome evolution .

backgroundpiriformis syndrome ( ps ) is an uncommon disease characterized by symptoms resulting from compression / irritation of the sciatic nerve by the piriformis muscle . uncertainty and controversy remain regarding the proper diagnosis and most effective form of treatment for ps . this study analyzes the diagnostic methods and efficacy of conservative and surgical treatments for ps.methodsfrom march 2006 to february 2013 , we retrospectively reviewed 239 patients who were diagnosed with ps and screened them for eligibility according to our inclusion / exclusion criteria . all patients underwent various conservative treatments initially including activity modification , medications , physical therapy , local steroid injections into the piriformis muscle , and extracorporeal shock wave therapy for at least 3 months . we resected the piriformis muscle with / without neurolysis of the sciatic nerve in 12 patients who had intractable sciatica despite conservative treatment at least for 3 months . the average age of the patients ( 4 males and 8 females ) was 61 years ( range , 45 to 71 years ) . the average duration of symptoms before surgery was 22.1 months ( range , 4 to 72 months ) , and the mean follow - up period was 22.7 months ( range , 12 to 43 months ) . we evaluated the degree of pain and recorded the responses using a visual analog scale ( vas ) preoperatively and 3 days and 12 months postoperatively.resultsbuttock pain was more improved than sciatica with various conservative treatments . compared with preoperatively , the vas score was significantly decreased after the operation . overall , satisfactory results were obtained in 10 patients ( 83% ) after surgery.conclusionsps is thought to be an exclusively clinical diagnosis , and if the diagnosis is performed correctly , surgery can be a good treatment option in patients with refractory sciatica despite appropriate conservative treatments .

surgery was performed on 12 patients from among 239 patients who were diagnosed with ps using our inclusion / exclusion criteria ( table 1 ) . of these surgery was indicated when pain ( buttock pain or mainly sciatica ) was not relieved by conservative measures , including education for habitual position or physical activity change , medication , physical therapy , local steroid injections on the piriformis muscle , or extracorporeal shock wave therapy ( eswt ) for at least 3 months . twelve patients underwent surgery for ps and the average age of the patients ( 4 males and 8 females ) who underwent surgery was 61 years ( range , 45 to 71 years ) . the average duration of the symptoms before surgery was 22.1 months ( range , 4 to 72 months ) , and the mean follow - up period was 22.7 months ( range , 12 to 43 months ) . of the 12 patients who had piriformis muscle resection with / without neurolysis , 8 had underlying pathologies including spinal stenosis ; 5 had been managed by spinal block and 3 had undergone lumbar spinal surgery , but their symptoms had not been relieved . three patients had a long - time , occupation - related , habitual sitting position , and 1 patient had a history of sacral fracture . we evaluated buttock pain with / without sciatica using a visual analog scale ( vas ; 0 , no pain ; 10 , maximum pain ) and recorded the responses preoperatively , and at 3 days and 12 months postoperatively . kruskal - wallis and mann - whitney u - tests were used for post hoc analysis to compare changes in vas for pain over time . the gluteus maximus was divided in the direction of its fibers by blunt dissection , and the fascia lata was incised in continuity where it overlaid the trochanter ; we then removed the trochanteric bursa . the piriformis muscle was inserted into the posterior aspect of the greater trochanter as tendinous nature and was located above the obturator internus tendon ( fig . the sciatic nerve was explored and found to pass anteriorly to the piriformis muscle in all cases . additionally , we divided the tight piriformis tendon at the insertion site at its tendinous portion . the proximal portion of the muscle was retracted when the leg was internally rotated after its division . neurolysis around the sciatic nerve into the sciatic notch was performed in two cases of a severely adherent sciatic nerve . severely dilated and engorged epineurial vessels were found in two cases with intractable sciatica ( fig . after surgery , patient activity with the assistance of a cane was encouraged to relieve pain from the gluteal muscle repair . the diagnostic procedure involved a detailed physical examination , including a palpation test for tenderness over the origin ( sacroiliac joint ) or insertion of the short external rotators behind the trochanter ( fig . the clinical examination also included several provocation tests for pain and weakness on resisted abduction and external rotation of the thigh in a sitting position ( pace test ) , pain on forced passive internal rotation of the extended thigh ( freiberg 's test ) , and buttock and leg pain during passive straight leg raising ( lasgue 's sign ) . in addition to electromyography , various imaging studies including ct , mri , and ultrasonography were performed , and a steroid injection into the piriformis muscle was carried out for diagnostic treatment . to exclude sciatica caused by spinal problems , repeated patients who had greater than 50% relief of symptoms of sciatica after caudal block were excluded from having a diagnosis of ps . asymmetry of the piriformis muscle on ct or mri was considered a positive finding of ps . an electromyographic finding of radiculopathy was attributed to spinal root compression , not to ps ( fig . the gluteus maximus was divided in the direction of its fibers by blunt dissection , and the fascia lata was incised in continuity where it overlaid the trochanter ; we then removed the trochanteric bursa . the piriformis muscle was inserted into the posterior aspect of the greater trochanter as tendinous nature and was located above the obturator internus tendon ( fig . the sciatic nerve was explored and found to pass anteriorly to the piriformis muscle in all cases . additionally , we divided the tight piriformis tendon at the insertion site at its tendinous portion . the proximal portion of the muscle was retracted when the leg was internally rotated after its division . neurolysis around the sciatic nerve into the sciatic notch was performed in two cases of a severely adherent sciatic nerve . severely dilated and engorged epineurial vessels were found in two cases with intractable sciatica ( fig . after surgery , patient activity with the assistance of a cane was encouraged to relieve pain from the gluteal muscle repair . the diagnostic procedure involved a detailed physical examination , including a palpation test for tenderness over the origin ( sacroiliac joint ) or insertion of the short external rotators behind the trochanter ( fig . the clinical examination also included several provocation tests for pain and weakness on resisted abduction and external rotation of the thigh in a sitting position ( pace test ) , pain on forced passive internal rotation of the extended thigh ( freiberg 's test ) , and buttock and leg pain during passive straight leg raising ( lasgue 's sign ) . in addition to electromyography , various imaging studies including ct , mri , and ultrasonography were performed , and a steroid injection into the piriformis muscle was carried out for diagnostic treatment . to exclude sciatica caused by spinal problems , repeated patients who had greater than 50% relief of symptoms of sciatica after caudal block were excluded from having a diagnosis of ps . asymmetry of the piriformis muscle on ct or mri was considered a positive finding of ps . an electromyographic finding of radiculopathy was attributed to spinal root compression , not to ps ( fig . for the diagnosis of ps , physical examinations , including several provocative tests , radiographic studies , such as plain x - ray , ct or mri , emg , or an injection were performed . when a diagnosis of ps was suspected , various conservative treatments were initially performed in all patients and generally provided good results ( table 2 ) . of the 239 patients , 12 patients who were refractory to conservative treatment underwent surgical treatment.table 3 summarizes the clinical features and the results of surgical treatment . on physical examination , tenderness in the gluteal area , particularly on the iliac side of the sacroiliac joint , was detected in 10 patients ( 83% ) . pain provocative tests such as the freiberg 's and pace tests were positive in 7 patients ( 58% ) . electrodiagnostic testing showed no specific findings ( delayed h - reflex at the flexion adduction internal rotation position ) suggesting ps as opposed to other pathologies , such as lumbosacral radiculopathy , sciatic nerve palsy , or posterior cutaneous neuropathy of the thigh . asymmetry of the piriformis muscle or hyperintensity around the sciatic nerve on ct and mri was detected in only 5 patients ( 42% ) ( fig . three patients had occupations that involved sitting for a long duration , such as sewing or driving . of the 12 patients undergoing surgical resection of the piriformis muscle , neurolysis was performed in 2 patients due to a severely adherent or scarred sciatic nerve . engorged epineurial vessels around the sciatic nerve shrank spontaneously after resection of the piriformis muscle . the average length of the skin incision was 9.5 cm , and the average amount of postoperative bleeding was 24.5 ml . there were no postoperative complications including hematoma , infection , delayed wound healing , scar formation , and myositis ossificans . the average duration of hospitalization was 5.3 days ( range , 3 to 9 days ) . compared with preoperatively ( mean vas score , 9 ) , the vas scores significantly decreased both immediately after surgery ( mean vas score , 4 ) and at the 12-month follow - up ( mean vas score , 3.1 ) ( table 4 ) . persistent buttock pain after surgery was present in 3 patients . among them , 1 patient had symptom relief after 12 months . the diagnosis of ps is mostly elusive and remains controversial due to the lack of consistent objective diagnostic criteria . the differential diagnoses should include herniation of the nucleus pulposus ( hnp ) , myofascial pain , sacroiliitis , trochanteric bursitis , or any other sciatic nerve - impinging conditions . in this study , the diagnosis of ps could be established with the patient 's history , a careful physical examination , and a local injection of the piriformis muscle when no other etiological findings were identified on emg or imaging studies , including ct and mri ( fig . because lumbosacral hnp or spinal stenosis commonly occurs at the l4 - 5 or l5-s1 intervertebral space , it is important to determine whether the pain originates from the root or peripheral nerve.10 ) in patients with ps , symptoms of neurological claudication are rare , while pain aggravation by a position change or prolonged sitting is frequently observed in them . specific sensory changes in dermatomes or muscle weakness can help to exclude ps . based on our cases , we suggest that the three cardinal symptoms of ps are buttock pain , radiating pain to the posterior thigh above the knee , and pain aggravated by position changes or prolonged sitting . the piriformis muscle can be firm and hard to palpation from the greater sciatic notch to the posterior aspect of the greater trochanter . in physical examination , tenderness of the piriformis muscle ( 83% ) is the most consistent finding . although emg is often normal in patients with ps , continuous compression may result in abnormal spontaneous activity of the muscles innervated by the sciatic nerve , including a delay in the h - reflex with the affected leg in a flexed , adducted , and internally rotated position.11 ) in our study , electrodiagnostic testing was not helpful for the diagnosis of ps but useful in ruling out other causes with similar symptoms , such as lumbosacral radiculopathy . diagnostic imaging modalities , including ct , mri , and mrn , have been used in many studies to diagnose ps.12131415 ) however , these studies are limited to cases showing atypical anatomy , including asymmetry of the piriformis muscle or hyperintensity of the sciatic nerve ; these conditions accounted for only 5 of the 12 surgical patients ( 42% ) in our study . sayson et al.16 ) and barton17 ) found that preoperative mri failed to identify atypical anatomy that was found intraoperatively . mrn is a relatively new technique that was developed specifically to enhance the imaging of nerves . filler et al.1 ) used mrn to prospectively investigate 87 patients with sciatica - like pain in whom either standard testing had failed to yield a diagnosis or who had failed lumbar disc surgery ; 67% of this group was diagnosed with ps . however , tiel18 ) pointed out methodological and technical problems of mrn . some reports have suggested diagnostic criteria for ps.1920 ) recently , michel et al.21 ) proposed to use a clinical scoring system : ps can be considered probable with a score of 8 or more out of 12 points . however , it includes negative findings for spinal disease , such as no lower back pain , painless axial spinal palpation , negative lasgue 's maneuver , or absence of perineal irritation ( 4 points ) . a scoring system including negative findings for spinal problems can result in overdiagnosis of ps . in addition , the scoring system involves obscure physical tests that provoke buttock pain or sciatica by stretching or resisted contraction , and it does not include some diagnostic tests , such as local steroid injection , which is a widely used tool for establishing the diagnosis . they are comprised of 5 items : buttock pain with / without sciatica during prolonged sitting ; tenderness of the piriformis muscle ; positive provocative tests ; positive findings on ct or mri ; and pain relief with a local injection . additionally , caudal or epidural block was tried at least once before surgery in our study . we suggest to exclude a diagnosis of ps with any of the following findings : symptoms of neurologic claudication ; positive lasgue 's or straight leg raise test ; sensory changes on nerve root innervation ; radiculopathy on emg ; or an effective caudal or epidural block . the sciatic nerve compression by the piriformis muscle or surrounding fibrous bands is different from the nerve root compression of spinal origin . absent clinical findings due to nerve root compression is important in the diagnosis of ps . initial nonoperative treatment of ps includes medications ( nonsteroidal anti - inflammatory drugs , muscle relaxants , and other medications effective in neuropathic pain , such as pregabalin or gabapentin ) , physiotherapy , eswt , injections of local anesthetics and corticosteroids , and the more recently investigated option of botulinum neurotoxin injections.8 ) in our study , eswt was applied in patients with buttock pain more than twice with an interval of 1 week until pain subsides significantly . eswt was undertaken with 2,000 pulses each time at 1 week interval totaling 4,000 to 6,000 pulses . our clinical results demonstrated that the most effective modality in treatment of ps for reducing buttock pain was eswt . we had satisfactory clinical results after release of the piriformis muscle and neurolysis of sciatic nerve in patients with refractory sciatica that fail to respond successfully to conservative treatments . intraoperatively , identification of the piriformis muscle among short external rotator muscles and posterior retraction after complete resection of the muscle are important . careful dissection of fibrous tissues around the sciatic nerve is also essential to avoid damaging the nerve proper or the dilated vaso nervorum . deep gluteal syndrome ( dgs ) is a disease entity that is characterized by pain or dysesthesia in the buttock area , hip , or posterior thigh and/or radicular pain due to a nondiscogenic sciatic nerve entrapment in the subgluteal space.22 ) its main pathology is fibrous bands around the sciatic nerve formed by various pathological conditions , such as piriformis syndrome , obturator internus / gemellus syndrome , or ischiofemoral impingement . the causes of dgs include traumatic , iatrogenic , inflammatory / infectious , vascular , and gynecological processes , and tumors / pseudotumors . therefore , the treatment of dgs is decompression of the sciatic nerve via open or endoscopic surgery . it is not clear whether resection of the piriformis muscle has additional benefits over decompression of the sciatic nerve in ps . however , the recurrence of sciatic nerve adhesions can be avoided when the muscle is resected.1 ) endoscopic surgery for adhesiolysis of the sciatic nerve has several advantages over open methods , including less invasiveness and less postoperative pain.23 ) however , endoscopic sciatic nerve release is technically demanding and has limited efficacy for release of a severely adherent sciatic nerve . considering that no prospective , randomized trial has evaluated surgical treatment outcomes of ps , the important finding of our study was the significant decrease in symptoms after surgery . to achieve good results , the indications for surgery ( no response to physical therapy and at least one injection ) should be determined upon proper diagnosis ( based exclusively on clinical and spine evaluations ) . surgery is an important treatment option for unresolved ps because of its low morbidity and simplicity . to overcome the limitations of our work , a prospective study with a greater number of cases and a longer follow - up period should be performed to establish the gold standard methods for the diagnosis and treatment of ps ; furthermore , several modalities for diagnosis of ps should be developed . in conclusions , the diagnosis of ps is obscure and elusive , but a systematic approach is helpful . if a diagnosis is determined correctly , surgical treatment can be a good option in patients with refractory pain , particularly sciatica , despite application of appropriate conservative treatment modalities .

the epitaxial growth of monocrystalline semiconductors on metal nanostructures is interesting from both fundamental and applied perspectives . the realization of nanostructures with excellent interfaces and material properties that also have controlled optical resonances can be very challenging . here we report the synthesis and characterization of metal semiconductor core shell nanowires . we demonstrate a solution - phase route to obtain stable core shell metal cu2o nanowires with outstanding control over the resulting structure , in which the noble metal nanowire is used as the nucleation site for epitaxial growth of quasi - monocrystalline cu2o shells at room temperature in aqueous solution . we use x - ray and electron diffraction , high - resolution transmission electron microscopy , energy dispersive x - ray spectroscopy , photoluminescence spectroscopy , and absorption spectroscopy , as well as density functional theory calculations , to characterize the core shell nanowires and verify their structure . metal semiconductor core shell nanowires offer several potential advantages over thin film and traditional nanowire architectures as building blocks for photovoltaics , including efficient carrier collection in radial nanowire junctions and strong optical resonances that can be tuned to maximize absorption .

abstractbackground : studies examining the efficiency of drug - coated balloon ( dcb ) + bare metal stent ( bms ) compared with stents alone for de novo lesions have reported inconsistent results . the present comprehensive meta - analysis of randomized controlled trials ( rcts ) assessed and compared the clinical efficacy and safety of dcb + bms with those of stents alone for de novo coronary artery disease.methods:we formally searched electronic databases before september 2016 to identify potential studies . all rcts were eligible for inclusion if they compared dcb + bms with a control treatment ( drug - eluting stent [ des ] alone or bms alone ) in patients with de novo coronary artery disease.results:eleven rcts with a total of 2196 patients met the inclusion criteria were included in our meta - analysis . subgroup analysis indicated dcb plus bms was associated with poorer outcomes when compared with des alone in primary endpoint { ( in - segment late lumen loss [ lll ] : mean difference [ md ] , 0.19 ; 95% confidence interval [ ci ] , 0.060.32 ; p = 0.0042 ) and ( major adverse cardiovascular events [ maces ] : risk ratio [ rr ] , 1.88 ; 95% ci , 1.442.45 ; p < 0.0001)}. however , dcb + bms had nonsignificantly lower lll than bms alone ( in - segment lll : md , 0.14 ; 95% ci , 0.330.04 ; p = 0.24 ) , and was more advantageous in reducing mace incidence , with borderline significance ( maces : rr , 0.67 ; 95% ci , 0.450.99 ; p = 0.05).conclusions : in summary , the present results do not favor the dcb + bms strategy as an alternative therapeutic method to des implantation for de novo coronary artery lesions in percutaneous coronary intervention ( pci ) . additional well - designed large rcts with long - follow - up periods are required to clarify the inconsistent results .

recent evidences support using paclitaxel drug - coated balloon ( dcb ) catheters as a therapeutic method for de novo coronary lesions , in - stent restenosis ( isr ) , small coronary vessels , and coronary bifurcation lesions . dcb was designed to achieve comparable efficacy in neointimal proliferation through local drug delivery without requiring foreign body implantation or prolonged dual antiplatelet therapy ( dapt ) . the advantages of dcb include homogeneous and high concentration 's drug delivery to the entire vessel wall , absence of stent layer , and absence of the polymer that could lead to chronic inflammation . dcb is a promising device to overcome some limitations of des in percutaneous coronary intervention ( pci ) , such as isr , late and very late stent thrombosis , and risk of bleeding caused by prolonged dapt . although dcb has shown remarkable angiographic and clinical effects in coronary artery interventional therapy , it has some limitations in the treatment of de novo coronary lesions . elastic recoil and flow - limiting dissections may be the main reasons for therapy failure . as the lack of mechanical scaffolding provided by stent struts , the use of dcb may not be ideal for complex coronary lesions . therefore , a strategy combining dcb and bare metal stent ( bms ) is a potential solution to overcome these limitations . the more rapid endothelialization and shorter dapt duration of bms than des should be beneficial in certain scenarios . however , studies examining the efficiency of dcb + bms compared with stents alone for de novo lesions have yielded inconsistent results , and whether this strategy provides additional benefits remains unclear . hence , we conducted a comprehensive meta - analysis of randomized controlled trials ( rcts ) to assess and compare the clinical efficacy and safety of dcb + bms with those of stents alone for de novo coronary lesions . we comprehensively searched related papers in electronic databases ( pubmed , web of science , and the cochrane central register of controlled trials ) before september 2016 to identify potential rcts . paclitaxel - eluting balloon , drug - eluting balloon , and drug - coated balloon . ethical approval was not required due to that this is a systematic review and meta - analysis . all included studies were approved by the notified ethics committees and institutional review boards . and this study was performed in accordance with preferred reporting items for systematic reviews and meta - analyses ( prisma ) statement . studies met the following inclusion criteria were included in the meta - analysis : rcts of de novo coronary artery lesions intervention , dcb + bms as a treatment arm , and eligible angiographic and clinical outcome data obtained during follow - up . data abstraction was performed independently by 2 investigators ( lu and zhu ) , and discrepancies were resolved by consensus . the following features of each eligible study were extracted using a standardized form : study and patient characteristics , intervention procedures , and angiographic and clinical outcomes . the cochrane collaboration tool was used to methodologically assess the risk of bias to evaluate the quality of included trials . the following methodological domains were considered : random sequence generation , allocation concealment , blinding , drop - out rates ( incomplete outcome data ) , addressing incomplete outcome data , selective reporting , and other potential sources of bias . after assessment , the included study were labeled as low risk ( l ) , high risk ( h ) , or the primary endpoints were in - segment late lumen loss ( lll ) and major adverse cardiac events ( maces ) . the secondary endpoints were in - segment binary restenosis ( br ) , in - segment minimum lumen diameter ( mld ) , and target lesion revascularization ( tlr ) , myocardial infarction ( mi ) , and death . maces were defined as a composite of death , mi , and tlr . the most similar endpoint was used if data for mentioned endpoint were unavailable . we conducted the meta - analysis by using the cochrane program review manager ( v.5.0 ; oxford , england ) and stata software ( version 12.0 ; statcorp , college station , tx ) . according to the inverse variance fixed - effect model , categorical variables were calculated as the pooled risk ratio ( rr ) and 95% confidence intervals ( cis ) . continuous variables were presented as estimated mean difference ( md ) with a 95% ci . if i > 50% ( substantial and important heterogeneity ) , a random effect model was used for quantitative data synthesis , whereas a fixed model was adopted . begger funnel plots and egger tests were used to assess publication bias , with p < 0.05 as the threshold for statistical significance . we comprehensively searched related papers in electronic databases ( pubmed , web of science , and the cochrane central register of controlled trials ) before september 2016 to identify potential rcts . paclitaxel - eluting balloon , drug - eluting balloon , and drug - coated balloon . ethical approval was not required due to that this is a systematic review and meta - analysis . all included studies were approved by the notified ethics committees and institutional review boards . and this study was performed in accordance with preferred reporting items for systematic reviews and meta - analyses ( prisma ) statement . studies met the following inclusion criteria were included in the meta - analysis : rcts of de novo coronary artery lesions intervention , dcb + bms as a treatment arm , and eligible angiographic and clinical outcome data obtained during follow - up . data abstraction was performed independently by 2 investigators ( lu and zhu ) , and discrepancies were resolved by consensus . the following features of each eligible study were extracted using a standardized form : study and patient characteristics , intervention procedures , and angiographic and clinical outcomes . the cochrane collaboration tool was used to methodologically assess the risk of bias to evaluate the quality of included trials . the following methodological domains were considered : random sequence generation , allocation concealment , blinding , drop - out rates ( incomplete outcome data ) , addressing incomplete outcome data , selective reporting , and other potential sources of bias . after assessment , the included study were labeled as low risk ( l ) , high risk ( h ) , or unclear risk ( u ) . the primary endpoints were in - segment late lumen loss ( lll ) and major adverse cardiac events ( maces ) . the secondary endpoints were in - segment binary restenosis ( br ) , in - segment minimum lumen diameter ( mld ) , and target lesion revascularization ( tlr ) , myocardial infarction ( mi ) , and death . maces were defined as a composite of death , mi , and tlr . the most similar endpoint was used if data for mentioned endpoint were unavailable . we conducted the meta - analysis by using the cochrane program review manager ( v.5.0 ; oxford , england ) and stata software ( version 12.0 ; statcorp , college station , tx ) . according to the inverse variance fixed - effect model , categorical variables were calculated as the pooled risk ratio ( rr ) and 95% confidence intervals ( cis ) . continuous variables were presented as estimated mean difference ( md ) with a 95% ci . if i > 50% ( substantial and important heterogeneity ) , a random effect model was used for quantitative data synthesis , whereas a fixed model was adopted . begger funnel plots and egger tests were used to assess publication bias , with p < 0.05 as the threshold for statistical significance . we initially screened a total of 7668 potential studies through a number of searches . after eliminating duplicates , 505 articles were examined . of these , 11 rcts with a total of 2196 patients met the inclusion criteria were included in our meta - analysis . figure 1 presents a flowchart of the overall search strategy . among these 11 studies , four studies were 3-arm trials comparing the subgroups dcb + bms , bms alone , and des alone ; therefore , these studies were considered as 2 separate trials . we finally selected 9 studies comparing dcb + bms with des alone and 6 comparing dcb + bms with bms alone . the clinical and angiographic primary endpoints were provided in all trials , with follow - up durations of 6 to 24 months . furthermore , dcb + bms was used in 714 patients , whereas control treatments , namely bms alone and des alone , were used in 190 and 715 patients , respectively . the key demographic and angiographic characteristics of included the studies are summarized in tables 1 and 2 , respectively . flow diagram for identification processes . general characteristics of studies included in this meta - analysis . lesions and devices characteristics of included studies . lll : this was reported in 9 of the 11 studies within follow - up periods of 6 to 9 months . nine studies were included in the dcb + bms versus des subgroup analysis , whereas 5 studies were included in the dcb + bms versus bms subgroup analysis . compared with the des alone subgroup , the dcb + bms subgroup exhibited a significant increase in lll ( md , 0.19 ; 95% ci , 0.060.32 ; p = 0.0042 ) . however , the dcb + bms subgroup showed nonsignificantly lower lll than did the bms alone subgroup ( md , 0.14 ; 95% ci , 0.330.04 ; p = 0.24 ; fig . effectiveness of dcb + bms strategy versus des alone or maces : these were observed in 10 of the 11 studies within a follow - up period of 6 to 24 months . the fixed effect model was used . subgroup analysis indicated that compared with des alone , dcb + bms significantly increased maces ( rr , 1.88 ; 95% ci , 1.442.45 ; p < 0.0001 ) . the subgroup analysis showed that the dcb + bms strategy was advantageous over the bms treatment in reducing maces incidence , with borderline significant ( rr , 0.67 ; 95% ci , 0.450.99 ; p = 0.05 ; fig . seven and 3 studies with follow - up periods of 6 to 9 months were included in the dcb + bms versus des alone and dcb + bms versus bms alone subgroup analyses , respectively . subgroup analysis showed the dcb + bms strategy was inferior to des alone strategy in reducing br incidence ( rr , 2.15 ; 95% ci , 1.074.31 , p = 0.03 ) . the dcb + bms versus bms subgroup analysis showed that dcb + bms was beneficial , but the difference between both strategies was nonsignificant ( rr , 0.74 ; 95% ci , 0.341.60 , p = 0.44 , respectively ; fig . effectiveness of dcb + bms strategy versus des alone or secondary angiographic endpoints : ( a ) in - segment binary restenosis rate ; and ( b ) in - segment minimum lumen diameter . six and 3 studies with follow - up periods of 6 to 9 months were included in the dcb + bms versus des alone and dcb + bms versus bms alone subgroup analyses , respectively . compared with des alone , dcb + bms had a significant lower mld ( md , 0.25 ; 95% ci , 0.41 to 0.10 ; p = 0.001 ) . a significant effect favoring dcb + bms was detected in the dcb + bms versus bms alone subgroup analysis ( md , 0.18 ; 95% ci , 0.030.33 ; p = 0.02 ; fig . all 3 endpoints were reported in 9 of the 11 studies within follow - up periods of 6 to 24 months . because of the low degree of heterogeneity , we used the fixed effect model for the quantitative analysis . tlr : the analysis indicated a significantly higher risk of tlr in the dcb + bms subgroup than in the des alone subgroup ( rr , 1.94 ; 95% ci , 1.272.98 ; p = 0.002 ) , and the incidence rate of tlr did not differ significantly between the dcb + bms subgroup and bms alone subgroup ( rr , 0.71 ; 95% ci , 0.471.09 ; p = 0.012 ; fig . mi : the analysis showed no significant difference in mi incidence between the dcb + bms and des alone subgroups ( rr , 0.88 ; 95% ci , 0.322.42 ; p = 0.81 ) . similarly , the incidence rate of mi was comparable following dcb + bms and bms alone implantation ( rr , 0.51 ; 95% ci , 0.161.67 ; p = 0.27 ; fig . death : the analysis revealed that death did not differ significantly in the dcb + bms and des subgroups ( rr , 5.91 ; 95% ci , 0.7248.39 ; p = 0.10 ) ; similar results were observed in the dcb + bms versus bms subgroup analysis ( rr , 0.20 ; 95% ci , 0.021.70 ; p = 0.14 ) . effectiveness of dcb + bms strategy versus des alone or secondary clinical endpoints : ( a ) target lesion revascularization , ( b ) mi , and ( c ) death . according to the results of heterogeneity analysis , we conducted sensitivity analysis between the dcb + bms and control groups ( dcb + bms vs des and dcb + bms vs bms subgroups ) at all observed endpoints . we sequentially eliminated one study at a time and observed that no study strongly influenced the overall results . egger test showed no evidence of significant publication bias in this meta - analysis ( p > 0.05 ) . in addition , the funnel plot was symmetrical , suggesting no publication bias ( fig . seven and 5 of the included studies showed a low risk of bias in random sequence generation and allocation concealment , respectively . five studies showed a low risk of bias in the blinding of participants , and 5 had a high risk of bias in the blinding of the outcome assessment . all studies have a low risk of bias regarding incomplete outcome data and selective outcome reporting . we initially screened a total of 7668 potential studies through a number of searches . after eliminating duplicates , 505 articles were examined . of these , 11 rcts with a total of 2196 patients met the inclusion criteria were included in our meta - analysis . figure 1 presents a flowchart of the overall search strategy . among these 11 studies , four studies were 3-arm trials comparing the subgroups dcb + bms , bms alone , and des alone ; therefore , these studies were considered as 2 separate trials . we finally selected 9 studies comparing dcb + bms with des alone and 6 comparing dcb + bms with bms alone . the clinical and angiographic primary endpoints were provided in all trials , with follow - up durations of 6 to 24 months . furthermore , dcb + bms was used in 714 patients , whereas control treatments , namely bms alone and des alone , were used in 190 and 715 patients , respectively . the key demographic and angiographic characteristics of included the studies are summarized in tables 1 and 2 , respectively . flow diagram for identification processes . general characteristics of studies included in this meta - analysis . lesions and devices characteristics of included studies . lll : this was reported in 9 of the 11 studies within follow - up periods of 6 to 9 months . nine studies were included in the dcb + bms versus des subgroup analysis , whereas 5 studies were included in the dcb + bms versus bms subgroup analysis . compared with the des alone subgroup , the dcb + bms subgroup exhibited a significant increase in lll ( md , 0.19 ; 95% ci , 0.060.32 ; p = 0.0042 ) . however , the dcb + bms subgroup showed nonsignificantly lower lll than did the bms alone subgroup ( md , 0.14 ; 95% ci , 0.330.04 ; p = 0.24 ; fig . effectiveness of dcb + bms strategy versus des alone or maces : these were observed in 10 of the 11 studies within a follow - up period of 6 to 24 months . the fixed effect model was used . subgroup analysis indicated that compared with des alone , dcb + bms significantly increased maces ( rr , 1.88 ; 95% ci , 1.442.45 ; p < 0.0001 ) . the subgroup analysis showed that the dcb + bms strategy was advantageous over the bms treatment in reducing maces incidence , with borderline significant ( rr , 0.67 ; 95% ci , 0.450.99 ; p = 0.05 ; fig . in - segment br rate . seven and 3 studies with follow - up periods of 6 to 9 months were included in the dcb + bms versus des alone and dcb + bms versus bms alone subgroup analyses , respectively . subgroup analysis showed the dcb + bms strategy was inferior to des alone strategy in reducing br incidence ( rr , 2.15 ; 95% ci , 1.074.31 , p = 0.03 ) . the dcb + bms versus bms subgroup analysis showed that dcb + bms was beneficial , but the difference between both strategies was nonsignificant ( rr , 0.74 ; 95% ci , 0.341.60 , p = 0.44 , respectively ; fig . effectiveness of dcb + bms strategy versus des alone or secondary angiographic endpoints : ( a ) in - segment binary restenosis rate ; and ( b ) in - segment minimum lumen diameter . in - segment mld . six and 3 studies with follow - up periods of 6 to 9 months were included in the dcb + bms versus des alone and dcb + bms versus bms alone subgroup analyses , respectively . compared with des alone , dcb + bms had a significant lower mld ( md , 0.25 ; 95% ci , 0.41 to 0.10 ; p = 0.001 ) . a significant effect favoring dcb + bms was detected in the dcb + bms versus bms alone subgroup analysis ( md , 0.18 ; 95% ci , 0.030.33 ; p = 0.02 ; fig . all 3 endpoints were reported in 9 of the 11 studies within follow - up periods of 6 to 24 months . because of the low degree of heterogeneity , we used the fixed effect model for the quantitative analysis . tlr : the analysis indicated a significantly higher risk of tlr in the dcb + bms subgroup than in the des alone subgroup ( rr , 1.94 ; 95% ci , 1.272.98 ; p = 0.002 ) , and the incidence rate of tlr did not differ significantly between the dcb + bms subgroup and bms alone subgroup ( rr , 0.71 ; 95% ci , 0.471.09 ; p = 0.012 ; fig . mi : the analysis showed no significant difference in mi incidence between the dcb + bms and des alone subgroups ( rr , 0.88 ; 95% ci , 0.322.42 ; p = 0.81 ) . similarly , the incidence rate of mi was comparable following dcb + bms and bms alone implantation ( rr , 0.51 ; 95% ci , 0.161.67 ; p = 0.27 ; fig . death : the analysis revealed that death did not differ significantly in the dcb + bms and des subgroups ( rr , 5.91 ; 95% ci , 0.7248.39 ; p = 0.10 ) ; similar results were observed in the dcb + bms versus bms subgroup analysis ( rr , 0.20 ; 95% ci , 0.021.70 ; p = 0.14 ) . effectiveness of dcb + bms strategy versus des alone or secondary clinical endpoints : ( a ) target lesion revascularization , ( b ) mi , and ( c ) death . according to the results of heterogeneity analysis , we conducted sensitivity analysis between the dcb + bms and control groups ( dcb + bms vs des and dcb + bms vs bms subgroups ) at all observed endpoints . we sequentially eliminated one study at a time and observed that no study strongly influenced the overall results . egger test showed no evidence of significant publication bias in this meta - analysis ( p > 0.05 ) . in addition , the funnel plot was symmetrical , suggesting no publication bias ( fig . 5 ) . funnel plot for publication bias . ( a ) primary angiographic endpoint : in - segment late lumen loss . ( b ) seven and 5 of the included studies showed a low risk of bias in random sequence generation and allocation concealment , respectively . five studies showed a low risk of bias in the blinding of participants , and 5 had a high risk of bias in the blinding of the outcome assessment . all studies have a low risk of bias regarding incomplete outcome data and selective outcome reporting . our present meta - analysis included the largest number of rcts to date showed that although the dcb + bms strategy performed more favorably than did the bms alone strategy , it was not superior to des alone strategy in the treatment of de novo coronary lesions . its dramatic ability to inhibit neointimal hyperplasia through sustained elution of cytostatic drugs turns into a significantly reduced repeat revascularization rate in clinical trials . nevertheless , cases of treatment failure , mainly because of isr and stent thrombosis ( st ) , have attracted more attention considering the sizeable number of patients with des implantation . various factors are required to satisfactorily resolve , such as slow drug release , polymer - induced inflammation , endothelial dysfunction , and coronary vasoconstriction disturbance . therefore , paclitaxel dcb may be an emerging therapeutic alternative that has the advantages of operative simplicity and homogeneous antiproliferative agent release along the entire device . to avoid the disadvantages of des , researchers have tried to combine dcb and bms to achieve benefits by dcb provided local release antiproliferative agents and bms prevented acute postangioplasty recoil . although bello study showed that , compared with pes in small vessels ( reference diameter 2.8 mm ) , dcb yielded significantly lower in - stent ( in - balloon ) late loss and similar rates of restenosis and revascularization . however , there have been few well - designed head to head studies comparing the dcb and des strategies for lesions with lumen diameters of more than 2.5 mm . all studies included in the present meta - analysis had applied the dcb + bms therapeutic strategy for de novo coronary lesions ( lumen diameter > 2.5 mm ) . nevertheless , the pooled results of our research showed that the clinical efficacy and safety of the dcb + bms strategy were not equivalent to those of the des alone strategy for de novo coronary lesions . regarding the maces rate , replacing des implantation with dcb + bms was not beneficial in simple de novo coronary lesion intervention . first , the lack of sufficient uncoated balloon predilation in some included study may have contributed to the result . predilation before dcb use could improve drug uptake by the vessel wall because of the creation of microdissections , thus facilitating drug transport through the intima and media , particularly for calcified lesions . the valentines ii trial adopted regular balloon predilatation of the target lesion followed dior ii dcb reported low in - segment lll and tlr rates . meanwhile , 1 rct , which adopted regular balloon predilatation , compared the efficacy of bms and dcb combination versus bms alone in patients with non - st elevation acute coronary syndrome also reported significantly lower lll but the absence of a favorable effect on patient clinical outcomes . second , we speculated geographical miss caused by unfavorable geometric proportions as a potential influencing factor because the reference point for stent or balloon placement was missing . one clinical trial reported that patients treated with dcb predilatation with an additional bms implantation had a very high proportion of geographical miss , which was identified as an independent significant predictor of restenosis . if stent deployment precedes dcb dilatation , the contact surface between the balloon and vessel wall is reduced by approximately 15% owing to the surface of the stent struts . the octopus trial , which used optical coherence tomography , reported that dcb + bms was associated with more pronounced neointimal proliferation than des . the ivus study used intravascular ultrasound also showed more pronounced neointimal hyperplasia in the dcb + bms group , leading to more revascularization than that in the des group . possible influencing factors are the interaction of the mounted stent with drug release from dcb , stent and balloon lengths , drug concentrations , and stent system . our meta - analysis included 2 strategies for dcb application : pre- and post - bms implantation . theoretically , dcb used before bms implantation could increase the risk of geographical mismatch , because the stent may be implanted partly outside the dcb - treated segment . by contrast , dcb used after bms implantation might affect the drug delivery to the vessel because of interposition of the stent struts . an optical coherence tomography ( oct ) study investigated the effects of the sequence of dcb and bms ( i.e. , dcb first and bms first ) and stated that the bms - first sequence translated into more favorable apposition than did the dcb - first sequence , as evidenced by the significantly low proportion of incomplete stent apposition ( isa ) struts and nonsignificantly low isa areas and volumes in the former . however , the indicor trial and another oct study used dcb from different manufacturers suggested that , the sequence of dcb application does not affect lll , maces , and in - stent neointimal hyperplasia . similar clinical and angiographic results were reported by the in - pact coro trial . finally , a possible explanation for these findings is that the currently used dcb , particularly first - generation dcbs , failed to warrant sufficient bioavailability of paclitaxel at the lesion site . bondesson et al reported the differential treatment outcomes of various dcbs , and this variation may be even larger than that caused by des because drug delivery to the vessel wall is crucial during balloon inflation . regarding lll , the pharmacokinetics of paclitaxel with first - generation dcbs may have been insufficient to provide comparable benefits . a recent experimental study showed much higher drug concentrations into the vessel wall by using the dior - ii dcb than dior - i , combined with a shorter inflation time . hence , using a second - generation dcb with a bms , higher tissue drug delivery dose , might lead to better angiographic and clinical outcomes for de novo lesions . second , because the studies had a relatively short follow - up durations , definitive conclusions will necessitate clinical follow - up for several additional years . finally , most included studies were conducted in western countries , hence , data from non - western countries were inadequate to precisely assess the clinical efficacy and safety of the dcb + bms strategy for de novo lesions . thus , further large , multicenter , well - designed randomized trials recruiting patients from more countries are required to provide additional insights . the present meta - analysis does not favor the dcb + bms strategy as an alternative therapeutic method to des implantation for de novo coronary artery lesions in pci . additional well - designed large rcts with long follow - up periods are required to resolve this concern .

background and objectives : mobilization of the colon and dissection of the mesentery are difficult laparoscopic techniques . traditional methods have been used for this dissection , but often with great difficulty . the ultrasonically activated shears , when introduced in 1993 , had the possibility to make this dissection less technically difficult . this is a retrospective review of the use of these shears for these techniques during laparoscopic - assisted colectomy.materials and methods : eighty - five patients underwent a laparoscopic - assisted right hemicolectomy or sigmoid resection . colon mobilization and mesenteric dissection were completed intracorporeally . complications , operative time , estimated blood loss , and length of stay were compared for resections completed with and without the ultrasonically activated shears.results:thirty-six patients had laparoscopic - assisted colectomy without the shears , and 49 patients had the procedure with the shears . there were no complications due to the ultrasonic energy . use of the shears resulted in shorter operative times ( 170 min . vs. 187 min . , p=0.1989 ) , similar median blood loss ( 98 ml vs. 95 ml , p=0.7620 ) , and shorter lengths of stay ( 4.3 days vs. 6.9 days , p=0.0018).conclusions : the ultrasonically activated shears are safe and effective for colon mobilization and mesenteric division . the use of the shears may result in shorter operative times and shorter lengths of stay .

minimally invasive surgical techniques have revolutionized the treatment of many of the problems seen by the general surgeon . although the impact has been greatest in the treatment of cholelithiasis , many of the same advantages achieved with laparoscopic cholecystectomy can be realized with advanced laparoscopic procedures . since the first initial reports of laparoscopic right hemicolectomy in february 1990 and sigmoid resection in october 1990 , laparoscopic - assisted colectomy ( lac ) has been found to have numerous advantages when compared to open colectomy . among these advantages are less blood loss , fewer wound complications , more rapid return of intestinal function , less pain , shorter hospitalization and quicker return to work . but lac has not been widely accepted as the surgical treatment of choice for patients requiring colon resection . first , the procedure is technically much more difficult and , second , although some have reported good results , lac has not yet been proven to yield equal or better results for the treatment of colon cancer when compared with open colectomy . indeed , much concern has been raised about the possibility of increased recurrence rates , port site metastasis , and the possibility that lac will not prove to be an adequate resection for cure of cancer . even though lac for benign disease has yielded good results , only a small percentage of surgeons offer lac for the treatment of benign disease when discussing options with their patients . overall , the biggest impediment to the widespread adoption of lac for benign disease remains the difficulty of the procedure . in our experience , colon mobilization and division of the mesentery have been the most difficult parts of the procedure for the surgeon to learn . the anastomosis is usually completed extracorporeally or with the transanal circular stapler , much as would be done during open surgery . the development of the ultrasonically activated shears ( laparosonic coagulating shears [ lcs ] , ethicon endo - surgery / ultracision , smithfield , ri ) has provided an alternative technology for mobilization of the colon and division of the mesentery . to evaluate the efficacy , safety , and efficiency of this new energy source , we retrospectively reviewed a portion of our series of lac cases . from october 1990 , to may 1997 , 118 laparoscopic colon resections were completed for a variety of indications . thirty - three of these patients had a colectomy other than a right hemicolectomy or a sigmoid resection and were eliminated from the study . the charts of the remaining 85 patients who underwent either laparoscopic - assisted right hemicolectomy or laparoscopic - assisted sigmoidectomy were reviewed retrospectively by the authors . patients who underwent curative resection for carcinoma of the colon were entered in an institutional review board ( irb ) approved prospective study . the operative notes were reviewed to determine the method in which the colon was mobilized and the mesentery divided . from this review , two groups were identified : one in which this dissection was done without the lcs ( no lcs group ) , and one in which this dissection was done with the lcs ( lcs group ) . the age , sex , indication for surgery , operative times , estimated blood loss ( ebl ) , and length of stay ( los ) were documented for each group . all surgical procedures were performed in a similar fashion by the senior author or by the laparoscopic surgery fellow under the direct supervision of the senior author . in all patients the colon was mobilized and the mesentery was divided with a totally laparoscopic technique . in cases completed without the lcs , hemostasis was obtained with a combination of clips , endoscopie linear cutting staplers , and monopolar cautery delivered with scissors . the technique using the lcs varied according to whether there was benign or malignant disease . in most cases of benign disease , other methods for hemostasis were usually not necessary unless the benign disease was so extensive that it required a wide dissection of the mesentery . for cases of malignancy , a high ligation of the vascular pedicle ( ileocolic artery for right colectomy and inferior mesenteric or superior sigmoid artery for sigmoid resection ) was accomplished with an endoscopie linear cutting stapler . a plastic wound protector was routinely placed in the mini - laparotomy incision during specimen extraction . the anastomosis after right hemicolectomy was completed extracorporeally via the minilaparotomy , and the anastomosis after sigmoid resection was created intracorporeally using the circular stapler passed transanally . all surgical procedures were performed in a similar fashion by the senior author or by the laparoscopic surgery fellow under the direct supervision of the senior author . in all patients the colon was mobilized and the mesentery was divided with a totally laparoscopic technique . in cases completed without the lcs , hemostasis was obtained with a combination of clips , endoscopie linear cutting staplers , and monopolar cautery delivered with scissors . the technique using the lcs varied according to whether there was benign or malignant disease . in most cases of benign disease , other methods for hemostasis were usually not necessary unless the benign disease was so extensive that it required a wide dissection of the mesentery . for cases of malignancy , a high ligation of the vascular pedicle ( ileocolic artery for right colectomy and inferior mesenteric or superior sigmoid artery for sigmoid resection ) was accomplished with an endoscopie linear cutting stapler . a plastic wound protector was routinely placed in the mini - laparotomy incision during specimen extraction . the anastomosis after right hemicolectomy was completed extracorporeally via the minilaparotomy , and the anastomosis after sigmoid resection was created intracorporeally using the circular stapler passed transanally . of the 85 patients , 36 had their procedures completed without the lcs and were in the no lcs group , while 49 had use of the lcs and were in the lcs group . the female : male ratio was 2:1 in the no lcs group and 1.6:1 in the lcs group . the average age was 67.9 years ( range 28 - 101 ) in the no lcs group and 62.6 years ( range 25 - 91 ) in the lcs group . right hemicolectomy was indicated for carcinoma 74% of the time in both groups ( table 1 ) . large adenomas , arteriovenous malformations , and , in one case , lymphoma were the other indications . sigmoid colectomy was indicated for diverticulitis in 58% of the no lcs group and 79% of the lcs group ( table 2 ) . the other indications for sigmoid colectomy in the no lcs group were sigmoid stricture and sigmoid volvulus . hence , the majority of the sigmoid resections were completed for diverticulitis , and the majority of the right hemicolectomies were for carcinoma . fifteen of 36 patients ( 42% ) in the no lcs group and 22 of 49 patients ( 45% ) in the lcs group had previous abdominal or pelvic surgery . average operating room time was less when the lcs was used ( 170 min . vs. 187 min . average blood loss was nearly the same whether the lcs was used or not ( 95 ml vs. 98 ml , p=0.7620 ) ( table 3 ) . the los was less for the lcs group ( 4.3 days vs. 6.9 days , p=0.0018 ) , and this did reach statistical significance . , three patients overall had postoperative bleeding from a stapled anastomosis , for an incidence of 3.5% . another patient after sigmoidectomy had an unsuccessful colonoscopic attempt to control the bleeding and required transanal suture of the staple line after 3 units of blood were transfused . a third patient bled from a stapled ileocolic anastomosis after heparin therapy was started to treat a postoperative pulmonary embolus . however , when heparin was again started , the patient rebled , and , therefore , a vena caval filter was placed . of note was that none of these patients who bled , bled from the area of dissection with the lcs , even when heparin therapy caused anastomotic bleeding . hence , there were no early or late bleeding complications in the areas of dissection with the lcs . additionally , there were no other early or late complications which could be related to the use of the shears . of the 85 patients , 36 had their procedures completed without the lcs and were in the no lcs group , while 49 had use of the lcs and were in the lcs group . the female : male ratio was 2:1 in the no lcs group and 1.6:1 in the lcs group . the average age was 67.9 years ( range 28 - 101 ) in the no lcs group and 62.6 years ( range 25 - 91 ) in the lcs group . right hemicolectomy was indicated for carcinoma 74% of the time in both groups ( table 1 ) . large adenomas , arteriovenous malformations , and , in one case , lymphoma were the other indications . sigmoid colectomy was indicated for diverticulitis in 58% of the no lcs group and 79% of the lcs group ( table 2 ) . the other indications for sigmoid colectomy in the no lcs group were sigmoid stricture and sigmoid volvulus . hence , the majority of the sigmoid resections were completed for diverticulitis , and the majority of the right hemicolectomies were for carcinoma . fifteen of 36 patients ( 42% ) in the no lcs group and 22 of 49 patients ( 45% ) in the lcs group had previous abdominal or pelvic surgery . average operating room time was less when the lcs was used ( 170 min . vs. 187 min . , p=0.1989 ) but did not reach statistical significance . average blood loss was nearly the same whether the lcs was used or not ( 95 ml vs. 98 ml , p=0.7620 ) ( table 3 ) . the los was less for the lcs group ( 4.3 days vs. 6.9 days , p=0.0018 ) , and this did reach statistical significance . , three patients overall had postoperative bleeding from a stapled anastomosis , for an incidence of 3.5% . another patient after sigmoidectomy had an unsuccessful colonoscopic attempt to control the bleeding and required transanal suture of the staple line after 3 units of blood were transfused . a third patient bled from a stapled ileocolic anastomosis after heparin therapy was started to treat a postoperative pulmonary embolus . however , when heparin was again started , the patient rebled , and , therefore , a vena caval filter was placed . of note was that none of these patients who bled , bled from the area of dissection with the lcs , even when heparin therapy caused anastomotic bleeding . hence , there were no early or late bleeding complications in the areas of dissection with the lcs . additionally , there were no other early or late complications which could be related to the use of the shears . this report is a single - institution , single - surgeon 's experience with lac , and dates from the first reported cases of lac . although many new instruments and technologies have been introduced since then , the fundamental surgical techniques and principles described then have not changed . the most difficult steps in lac are intracorporeal mobilization of the colon and division of the mesentery . the learning curve for these techniques is much longer than for the techniques required for laparoscopic cholecystectomy , and this has slowed the widespread use of lac for patients needing colon resection . this study was done to assess the use of a new technology , the ultrasonically activated shears , for mobilization of the colon and division of the mesentery . the jaws of the shears consist of an active blade and an opposing passive ( not ultrasonically activated ) , movable tissue pad . this allows the surgeon to grasp tissue and vessels within the jaws of the shears , and coapt the endothelium of any vessels in the tissue . the ultrasonic energy is then transmitted to this tissue and can seal blood vessels and divide what has been grasped . the shears have been shown to facilitate completion of other advanced laparoscopic procedures such as division of the short gastric arteries during nissen fundoplication and division of the infundibulopelvic ligament during laparoscopic - assisted vaginal hysterectomy . depending on the power setting of the generator , the active blade will move 50 - 100 microns with each oscillation . touching the active blade to tissue transfers mechanical energy from the blade to the tissue . this mechanical energy breaks hydrogen bonds in the protein of the tissue , resulting in a sticky coagulum which seals blood vessels . this will allow blood vessels up to 3 mm to be sealed with the shears , without the need for any other method to achieve hemostasis . relatively little heat is generated compared to other energy sources , since most of the energy delivered is mechanical energy . the relatively low level of heat generated increases the safety with which the instrument can be used adjacent to other viscera , such as the small intestine or great vessels . the largest or named arteries in the mesentery may need to be controlled by other means , such as clips , ligatures , or the endoscopie linear cutting stapler . we found that when the ultrasonically activated shears were utilized , the need for scissors , pre - tied loops , clips , and linear cutting staplers was markedly reduced . in situations in which blood vessels were less than 3 mm ( as in colon resections for benign disease when high vascular pedicle division was not necessary ) these were no longer needed , and the entire dissection could be completed with the shears . when we compared lac done with and without the shears , the overall operative times and blood loss were similar . although the operative times with the shears were a little bit shorter , this could have been due to an increase in our skills as we progressed along our learning curve . however , as our skills improved , and partly due to the availability of the shears , we attempted and completed many more difficult procedures than we would have tried without the shears , as documented by the 20% higher incidence of diverticulitis in the lcs group . these more difficult cases inevitably would have taken more time than most of the cases we tried initially if we had not had the shears . therefore , the shorter length of time in the group in which the shears were used , although not great , is probably significant since we were often doing more difficult cases with the shears . it is our opinion that use of the shears greatly facilitated successful completion of these more complex cases . the literature documents a decreased length of stay following lac . in the present study , although the los for the group in whom we used the shears was less , this difference is probably due to changes in our postoperative management as we became more comfortable and familiar with the recovery of patients after lac . since the patients treated without the shears were all treated early in our experience ( before the shears were available ) , the decrease in length of stay was probably related to our experience . with experience we learned that early advancement of the diet and earlier discharge are possible because the patient has less pain and a shorter ileus following lac . we do not see a reason why the use of the ultrasonically activated shears would reduce pain and shorten ileus , nor do we see a reason why the use of the lcs would explain the shorter length of stay . the ultrasonically activated shears are a safe and effective device for mobilizing the colon and dividing the mesentery during lac . for the experienced laparoscopic surgeon , use of the shears can reduce the time required for routine cases of lac and for the inexperienced laparoscopic surgeon , there is no substitute for appropriate training , but the shears have the potential to shorten the learning curve for the inexperienced surgeon by facilitating the two most difficult technical parts of lac .

summaryneurons in the mammalian neocortex arise from asymmetric divisions of progenitors residing in the ventricular zone . while in most progenitor divisions , the mitotic spindle is parallel to the ventricular surface , some progenitors reorient the spindle and divide in oblique orientations . here , we use conditional deletion and overexpression of mouse inscuteable ( minsc ) to analyze the relevance of spindle reorientation in cortical progenitors . mutating minsc almost abolishes oblique and vertical mitotic spindles , while minsc overexpression has the opposite effect . our data suggest that oblique divisions are essential for generating the correct numbers of neurons in all cortical layers . using clonal analysis , we demonstrate that spindle orientation affects the rate of indirect neurogenesis , a process where progenitors give rise to basal progenitors , which in turn divide symmetrically into two differentiating neurons . our results indicate that the orientation of progenitor cell divisions is important for correct lineage specification in the developing mammalian brain .

neurons arise from a small set of progenitor cells that divide in a spatially and temporally controlled manner to generate the six - layered structure of a fully functional adult cortex ( caviness et al . , 2009 ; gtz and huttner , 2005 ; pierani and wassef , 2009 ; rowitch and kriegstein , 2010 ) . how different fates are established in the daughter cells of these progenitors is poorly understood . during early phases of mouse brain development ( e9.0 ) , the cortex consists of neuroepithelial progenitors ( neps ) , which extend from the ventricular ( apical ) to the pial ( basal ) surface of the neural tube and divide symmetrically to amplify the progenitor pool . at the onset of neurogenesis ( around e11.0 ) , neps turn into so - called radial glial cells ( rgcs ) and adopt molecular and morphological characteristics of glial cells . rgcs are characterized by an apical fiber extending toward the ventricle and a basal fiber extending toward the pial surface ( caviness et al . , 2009 ; gtz and huttner , 2005 ; kriegstein and alvarez - buylla , 2009 ) . rgcs occupy the most apical area of the cortex , called the ventricular zone ( vz ) . their nuclei undergo a characteristic interkinetic nuclear migration where mitosis and s phase occur in the apical and basal areas of the vz , respectively . rgcs give rise to all the cortical neurons through two kinds of asymmetric divisions ( anthony et al . , they divide into one rgc and another cell that migrates into the more basally located cortical plate ( cp ) where it differentiates into a neuron . ipcs ( also called basal progenitors [ bps ] or nonsurface - dividing [ ns - div ] cells ) lose their connection to the apical surface and reside in the cortical area between the vz and intermediate zone ( iz ) where they form the so - called subventricular zone ( svz ) . ipcs undergo one to two rounds of symmetric division , generating either one or two pairs of neurons ( haubensak et al . 2004 ) , which can then populate all six layers of the cortex ( kowalczyk et al . , 2009 ; sessa et al . , 2008 ) . as the gene expression profiles of rgcs generating neurons are characteristically different from the ones generating ipcs ( pinto et al . , 2008 ) , the two modes of division seem to occur in distinct subpopulations of rgcs . whether or not the orientation of rgc divisions is relevant for neurogenesis has been a matter of intense debate . early reports have demonstrated that vertical spindle orientation correlates with an asymmetric outcome in terms of daughter cell fates ( chenn and mcconnell , 1995 ; zhong and chia , 2008 ) , leading to models in which the unequal segregation of the apical and basal plasma membranes directs cell fate ( zhong and chia , 2008 ) . consistent with this , mitotic spindles with vertical orientations are only found during the neurogenic phases of brain development ( haydar et al . , 2003 ) , while during the early expansion phase , keeping precise horizontal spindle orientation is crucial to maintain the neural progenitor pool ( fish et al . , 2006 ; yingling et al . , 2008 ) . the frequency of vertical divisions during the neurogenic phase , however , is too low to account for all divisions with asymmetric outcome ( chenn and mcconnell , 1995 ; haydar et al . , 2003 ; this could be explained by the small size of the apical membrane domain of rgcs , such that even barely oblique mitotic spindles would give rise to cleavage planes that fail to bisect this domain resulting in its asymmetric segregation ( kosodo et al . , 2004 ) . it has been demonstrated that increasing the rate of vertical divisions can affect progenitor cell number and location ( konno et al . , 2008 ; shitamukai et al . , 2011 ) . functional evidence to demonstrate that either vertical or oblique spindle orientation is required for neurogenesis , however , remains to be established . the molecular machinery for spindle orientation during neurogenesis is best understood in drosophila ( siller and doe , 2009 ) . in drosophila neuroblasts , orientation of the mitotic spindle along the apical - basal axis is important for the asymmetric segregation of the cell fate determinants numb ( hirata et al . , 1995 ; knoblich et al . , 1995 ; rhyu et al . , 1994 ; spana and doe , 1995 ) , prospero ( hirata et al . , 1995 ; knoblich et al . , 1995 ; spana and doe , 1995 ) , and brat ( bello et al . , 2006 ; betschinger et al . , 2006 ; lee et al . , 2006 ) into the basal daughter cell ( bowman et al . , 2006 ; 2006 ) , where these proteins prevent self - renewal and induce differentiation . in neuroblasts , the mitotic spindle is oriented by two protein complexes that assemble on its apical cell cortex . one complex consists of the pdz domain proteins par-3 , par-6 , and the atypical protein kinase c ( apkc ) while the other contains the goloco domain protein pins , the heterotrimeric g protein subunit gi , and the microtubule - binding protein mushroom body defect ( mud ) . , 2000 ; yu et al . , 2000 ) and par-3 ( schober et al . , 1999 ; wodarz et al . , 1999 ) via multiple armadillo repeats within its so - called asymmetry domain ( knoblich et al . in insc mutants , mitotic spindles in neuroblasts are randomly oriented , leading to missegregation of cell fate determinants , and thus , cell fate defects in the developing nervous system . when insc is ectopically expressed in epithelial cells , pins and mud are recruited from the basolateral to the apical cortex , and the mitotic spindle reorients from a horizontal into an apical - basal direction . therefore , unlike all other components , insc is not only required but also sufficient for spindle orientation along the apical - basal axis . while the components of the drosophila spindle orientation machinery are conserved in mammals , they have been studied mainly with regard to their roles in epithelial cell polarity ( goldstein and macara , 2007 ) , and most of them have additional functions in cell polarity or microtubule dynamics ( woodard et al . , 2010 ) . mammalian par-3 , par-6 , and apkc are important for spindle orientation , and like their drosophila counterparts they are also required for epithelial apical - basal polarity . pins has two mammalian homologs , ags-3 and lgn ( sanada and tsai , 2005 ; yu et al . , 2003 ) . ags-3 does not appear to be expressed in the brain at significant levels , and ags3 knockout mice show no brain phenotype ( blumer et al . , 2008 ) . by contrast , lgn mediates planar spindle orientation in the developing brain ( konno et al . , 2008 ; morin et al . , 2007 ) , consistent with its role in mitotic spindle orientation during epithelial morphogenesis ( zheng et al . , 2010 ) , but is also required for microtubule aster formation and spindle morphology ( du et al . , 2001 ) , and regulates mitotic spindle orientation during epithelial morphogenesis similarly , the mammalian mud homolog numa has been shown to play a conserved role in the spindle orientation complex ( du and macara , 2004 ) but has additional functions in organizing a bipolar mitotic spindle ( silk et al . , 2009 ; sun and schatten , 2006 ) . overexpression and rnai studies have shown that the protein is involved in orienting the mitotic spindle in the rat retina ( zigman et al . , 2005 ) , and a similar function has been postulated in the mouse skin ( lechler and fuchs , 2005 ) . moreover , in situ hybridization experiments showed that mouse inscuteable ( minsc ) is expressed in the developing neocortex at the time when the first neurons start to appear ( zigman et al . , 2005 ) . to test the role of minsc in cortical development , we measure spindle orientation in 3d and show that the fraction of oblique divisions increases or decreases upon minsc overexpression or deletion , respectively . we show that loss of minsc leads to defects in neurogenesis and depletion of bps , while minsc overexpression has the opposite effect . our data are consistent with a model in which oblique divisions preferentially give rise to bps and , therefore , suggest a mechanism regulating the balance between direct and indirect neurogenesis during mouse brain development . minsc is expressed throughout the developing cortex during mid - neurogenesis ( figures 1a , 1b , and 1k ) ( zigman et al . , 2005 ) . in the vz , the protein is enriched at the spindle midzone in about 90% of the anaphase cells ( yellow arrow in figure 1c , and graph in figure 1e ) . in 100% of the cp neurons , however , the protein is localized to the neuron cell body cytoplasm and concentrates on one side of the nucleus ( yellow arrow in figure 1d ) . to test whether minsc can functionally replace the drosophila protein , we generated transgenic flies expressing c - terminally myc - tagged minsc ( minsc::myc ) . when expressed in neuroblasts , minsc::myc localizes into an apical crescent ( figures 1f and 1 g ) . like drosophila insc ( kraut et al . , 1996 ) , minsc::myc can induce a reorientation of the mitotic spindle into an apical - basal orientation when ectopically expressed in epithelial cells ( figures 1h and 1i ) . thus , minsc is a functional homolog of drosophila insc . to analyze the function of minsc in mouse cortical development , we generated conditional loss - of - function and overexpression alleles ( called minsc and r26 , respectively ) ( figure 1j ; see figures s1a and s1b and supplemental experimental procedures available online for details ) . upon cre recombination , the r26 line lost -gal expression and showed strong and ubiquitous expression of minsc - gfp ( r26 ) ( figure 1p ) . for brain - specific recombination we used nescre8 , which expresses cre in the forebrain of e8.5 embryos ( petersen et al . , 2002 ) . when combined with minsc , this line results in near - complete removal of minsc from the cortex at e14.5 ( figures 1k and 1l ) . we call the recombined allele minsc . residual minsc staining on the apical surface of the cortex ( white arrow ) is presumably due to truncated protein persistence or mosaic expression of cre ( figure 1l ) . in addition we detected some nonspecific antibody staining around blood vessels ( yellow arrow ) that is not due to the secondary antibody ( figures s1f and 1 g ) . when combined with the r26 allele , nescre8 caused loss of -gal expression ( compare figure 1 m with 1n ) , and strong expression of the gfp fusion protein ( figures 1o and 1p ) in the entire cortex at e14.5 . expression of the gfp fusion protein can also be detected as a 90 kda band in immunoblots from e13.5 heads ( figure 1q ) . this band is found in addition to the 60 kda band from the endogenous protein in nescre/;r26 but not in nescre/ or r26 mice ( figure 1q ) . thus , we have generated functional tools for gain- and loss - of - function analysis of minsc . previous rnai experiments have suggested that the function of minsc in controlling the orientation of neural precursor divisions is conserved from drosophila to mice ( zigman et al . , 2005 ) . we therefore asked whether minsc controls the orientation of mitotic spindles in rgcs and , if so , whether the loss- and gain - of - function alleles influence spindle orientation and lineage specification in the developing brain . various conflicting reports exist on the wild - type orientation of mitotic spindles in rgcs ( chenn and mcconnell , 1995 ; haydar et al . , 2003 ; konno et al . , 2008 ) . in these reports , spindle orientations were measured relative to a line representing the ventricular surface . as this methodology neglects spindle orientations in z direction ( out of the focal plane ) and is therefore imprecise due to the curved apical surface of the ventricle , we used 3d image reconstruction and computational analysis to obtain more precise measurements . e11.5 and e13.5 embryos were stained for tubulin ( tub ) , tubulin ( tub ) , and phosphorylated histone h3 ( ph3 ) to mark centrosomes , mitotic spindles , and mitotic chromatin , respectively . embryonic brains were paraffin embedded , and individual anaphase rgcs were reconstructed in 3d from confocal stacks of coronal brain sections ( figures 2a2c ; figures s2a s2c ; asterisks in figures 2a and 2b point at centrosomes ) . using the imaris 3d visualization software , we then defined the position of the two centrosomes and placed five points at different positions along the apical surface of the 3d - rendered cell . these points were used to determine the best - fitting plane by orthogonal distance regression and to calculate the angle between a vector connecting the two dots marking the centrosomes , and the normal vector of the plane , marking the apical surface . the angle of the spindle orientation was calculated as 90 minus the angle ( figure 2d ) . using this procedure , we determined the division angle of radial glia cells from nescre/ ( ctrl ) , nescre/;minsc ( cko ) , and nescre/;r26 ( cki ) mice at both e11.5 and e13.5 . at e11.5 , rgcs divide in a planar orientation with mitotic spindles oriented in parallel to the ventricular surface ( angles less than 30 ) , consistent with previous observations ( haydar et al . , 2003 ; konno et al . division angles in cko and cki mice are not significantly different from controls ( figure 2e ; table s1 ) . although we can not exclude that cre recombination is not efficient in early stages , this suggests that minsc is not functional at early stages of neurogenesis . at e13.5 , however , 63% of the mitotic spindles in control embryos are at angles between 0 and 30 , while 33% are between 30 and 60. consistent with previous reports , we found that vertically oriented mitotic spindles ( between 60 and 90 ) are rare ( haydar et al . , 2003 ) and are not seen in more than 3% of all mitotic cells ( figure 2f , and blue bar in figure 2 g ) . in cko mice , however , the vast majority of mitotic spindles ( 95% ) were between 0 and 30 , oblique divisions ( 30 < 0 < 60 ) were strongly reduced ( 5% ) , and vertical spindles were never seen ( figure 2f , red bar in figure 2 g ; table s1 ) . upon overexpression of minsc in cki mice , however , the fraction of oblique and vertical divisions was significantly increased ( 63% ) ( figure 2f , green bar in figure 2 g ; table s1 ) . thus , like in drosophila , minsc is required and sufficient for orienting mitotic spindles along the apical - basal axis . importantly , the minsc spindle orientation phenotype is different from the one observed in lgn knockout mice and lgn knockdown in chicken spinal cord ( konno et al . , 2008 ; morin et al . , 2007 ) , where spindle orientation is randomized , and the number of horizontal spindles is actually decreased . we also tested the subcellular localization of minsc::gfp in r26/ rgcs at e14.5 ( figure 2h ) . when nuclei are close to the ventricular surface in preparation for mitosis , the protein concentrates in the apical stalk as well as on the basal side of the rgc body ( yellow arrow in figures 2h and 2i , and yellow bar in figure 2 m ) . in about 80% of mitotic cells , minsc concentrates in an apical crescent ( orange arrow in figures 2h and 2j , and green bar in figure 2 m ) that enlarges in prometaphase while the basal staining disappears ( green arrow in figures 2h and 2k ) . in anaphase , finally , minsc becomes symmetric again and is distributed on both sides of about 90% of anaphase rgcs and in the spindle midzone ( blue arrow in figures 2h and 2l , and blue bar in figure 2 m ) . taken together , these data demonstrate that minsc is an important regulator of spindle orientation in the developing mouse brain . both nescre/ ; minsc and nescre/;r26 mice survive to adulthood , although nescre/;r26 animals frequently show epileptic crisis . despite their viability , however , both mutants show clearly visible and reproducible defects in cortical organization ( figure 3 ) . nissl staining of brains from 2-month - old animals shows that cortical thickness is reduced in nescre/;minsc mice and increased in nescre/;r26 mice ( figures 3a3d ) . very similar brain defects are observed in minsc mice ( figure 3b ) , indicating that the time of onset of nescre8 expression is not relevant for the strength of the phenotype . the different layers of the developing cortex can be recognized by their unique cell density and morphology in nissl stains . analysis of the various minsc alleles reveals that layer organization is not dramatically affected in nescre/;minsc mice ( figure 3c ) while in nescre/;r26 mice , layer iv is barely recognizable and seems to be fused with layer v ( figure 3d ) . in addition , gfap staining indicates an alteration of the white matter layer thickness ( figure s3 ) . to further characterize these adult brain phenotypes , we used layer - specific markers . we used foxp2 as a marker for layer vi , foxp1 as a marker for layers iii and v , satb2 as a marker for layers ii iv ( britanova et al . 2003 ) , and cux1 as marker for layers iii iv ( nieto et al . , 2004 ) . the number of satb2-positive cells is reduced in the nescre/;minsc mice ( figures 3e and 3i ) , while in the nescre/;r26 mice , their number is increased ( figure 3 m ) . similar results were obtained using cux1 antibody as marker of the upper layers ( figures 3h , 3l , and 3p ) . the nuclear marker foxp1 is present in two stripes corresponding to layers iii and v ( figure 3f ) . cell density is strongly reduced in nescre/;minsc mice ( figure 3j ) while the number of cells in these layers is significantly increased in nescre/;r26 mice ( figure 3n ) . finally , layer vi of the adult cortex , which is the first to be formed during embryogenesis , is also affected in both mutant conditions , as shown by immunostaining with the foxp2 antibody ( figures 3 g , 3k , and 3o ) . a quantitative analysis of the cells positive for the various layer - specific markers ( figure 3q ) confirms this phenotypic analysis . these data demonstrate that minsc levels are important for neurogenesis and suggest that vertical orientation of the mitotic spindle is relevant for cortical development . to determine the developmental origin of those cortical phenotypes , we analyzed cortical development in minsc knockout and overexpression mice ( figure 4 ) . e11.5 brain sections from control , conditional knockout , germline - transmitted knockout , and conditional knockin show no difference in the expression of nestin , tbr1 ( neurons ) , or tbr2 ( intermediate progenitors ) ( figure s4 ) , indicating that minsc has no obvious role in early neurogenesis . we then stained coronal sections of e14.5 control and mutant brains with cresyl violet ( figures 4a4j ) . no major structural abnormalities were detected at this stage , but nescre/;minsc brains were smaller than controls ( figures 4a and 4b ) . the lateral ventricles were enlarged , and overall cortical thickness as well as the protrusion of the lateral ganglionic eminence into the ventricles were reduced ( figures 4a , 4b , 4e , and 4f ) . nescre/;r26 brains , on the other hand , exhibited increased cortical thickness ( figure 4a , 4c , 4e , and 4 g ) . these alterations in cortical thickness were observed in both central and lateral regions ( refer to the scheme in figure 4k ) . quantification of those phenotypes showed that cortical thickness was reduced by around 20% both medially and laterally in nescre/;minsc mice , while it was increased by about 20% in the medial region and by around 40% in the central and lateral regions in nescre/;r26 mice ( figure 4l ) . a more detailed examination of these phenotypes revealed that the alterations in cortical thickness are largely due to changes in the iz and the cp while the vz is almost unaffected ( figures 4h4j ) . in nescre/;minsc mice the thickness of the iz and cp were reduced by about 25% and 40% ( figure 4 m ) while in nescre/;r26 mice , both layers were thicker , up to more than three times ( figure 4 m ) . to test whether these alterations were due to changes in cellular composition , we stained e14.5 nescre/ , nescre/;minsc , and nescre/;r26 embryos for nestin , bplp , and tuj1 , which label neural progenitors and neurons , respectively ( menezes and luskin , 1994 ; yachnis et al . , 1993 ) . while nestin staining of nescre/;minsc mice does not reveal any obvious abnormalities , rgcs in nescre/;r26 brains showed an alteration in the radial organization of the rgc fibers ( menezes and luskin , 1994 ) ( figures 4n4p ; figure s5 ) . tuj1 staining revealed that neurons in the iz and cp of nescre/;minsc brains are reduced while in nescre/;r26 brains the area occupied by those neurons is enlarged , consistent with the observed increase in cortical thickness ( figures 4q4s ) . in nescre/;minsc brains , however , tuj1 neurons in the vz were rarely found ( arrow and inset in figure 4r ) , while in nescre/;r26 brains they seemed to be more abundant ( arrow and inset in figure 4s ) . together , these data indicate that changes in spindle orientation do affect neurogenesis in the developing cortex , with consequent alteration of its thickness , although the number of rgcs is not strongly affected ( figure 5r ) . to characterize the initial defects in minsc - deleted or -overexpressing mice , we used markers for cp neurons . cp neurons are the first recognizable layer of the developing neocortex , and are identified by the expression of map2 and the transcription factor tbr1 ( fujimori et al . , 2002 ; hevner et al . , the number of tbr1 cells is reduced by almost half in nescre/;minsc brains while in nescre/;r26 mice the number of these cells is significantly increased ( figures 5a5c and 5p ) . staining for map2 shows similar alterations in cp neurons in the two genotypes ( figures 5h5j ) . to test whether the decrease in neurogenesis occurs at the expense of cortical progenitor cells , we used the nuclear rgc marker pax6 ( figures 5d5f and 5l5n ) ( gtz et al . , 1998 ) . although the high density of pax6 nuclei in the vz makes it impossible to obtain precise quantitative measurements , we did not find any striking changes in the number of sox2 + vz progenitors in nescre/;minsc or in nescre/;r26 mice ( figure 5r ) . however , vertical spindle reorientation in nescre/;r26 mice leads to the frequent generation of pax6 progenitors that are located outside the vz , in the iz , or the cp ( figures 5f and 5n ) . the number and frequency of those cells were increased even more when cre recombination was induced in the germline of r26/ mothers using morecre . in e14.5 embryos from those mothers ( named r26/ ) , clusters of pax6 cells were frequently seen in the iz , and pax6 cells were present even in the cp where they replaced differentiating neurons forming gaps in the map2 layer of cells ( figures 5 g and 5k , arrows , and figure 5o ) . quantitative analysis revealed that pax6 cells were six times more abundant in the iz and three times more abundant in the cp of r26/ mice when compared to nescre/;r26 ( figure 5q ) . these observations are consistent with previous in utero electroporation experiments ( konno et al . , 2008 ) , although the previous conclusion that endogenous minsc does not orient mitotic spindles in the mouse cortex ( fish et al . , 2008 ; konno et al . , 2008 ) changes in cortical thickness and neuronal differentiation observed in minsc mutant and minsc - overexpressing brains could be due to alterations in the position of mitotic cells and/or in rgc proliferation . in order to distinguish between these possibilities , we first stained e14.5 sagittal brain sections with anti - ph3 to look at proliferative cells in both vz and svz . in control animals , 80% of the mitotic figures seen in the cortex at e14.5 are located at the apical side of the vz while 20% of mitotic figures corresponds to the more basally located intermediate progenitors ( figures 6a , 6d , 6h , and 6i ) . in nescre/;minsc mice , however , the number of basally located mitotic cells is strongly reduced at e14.5 ( figures 6b , 6e , 6h , and 6i ) . in nescre/;r26 mice , on the other hand , the number of basal mitotic cells is increased ( figures 6c , 6f6i ) . thus , changes in spindle orientation affect the number of basal mitotic cells , without significantly altering the number of apical mitotic cells ( figure 6h ) . alterations in the number of neurons and intermediate progenitors that are produced during neurogenesis could be due to premature cell cycle exit of vz progenitors . to test this , we injected pregnant females with brdu to label s phase cells , sacrificed the animals 24 hr hour later , and performed double immunostaining for brdu and the proliferation marker ki67 . in this experiment , the fraction of ki67brdu cells within the total brdu - positive population can be used as an indicator of cell cycle exit of progenitors . we found no significant differences in nescre/;minsc , in nescre/;r26 , or in r26/ mice ( figure 6j ) , indicating that minsc has no strong effect on average cell cycle length both in apical and bps . the altered proliferation pattern could be due to a difference in position or fate of the dividing cells . in wild - type animals , proliferation basal to the vz is due to ipcs , which can be specifically marked by staining for tbr2 ( figure 6k ) ( englund et al . , 2005 ) . in nescre/ ; minsc mice , the number of tbr2cells is reduced ( figure 6l ) while this number is increased in nescre/;r26 brains ( figure 6 m ) . this effect can be enhanced by germline recombination of the r26 allele in r26/ mice ( figure 6n ) . interestingly , the extra bps are no longer confined to the svz but frequently found in the more basal parts of the cortex . thus , modifying spindle orientation changes the frequency with which rgcs give rise to intermediate progenitors . as proliferation and cell cycle exit rates of rgcs do not change in the mutant conditions , we can exclude that this is a consequence of alterations in rgc proliferation . therefore , we postulate that spindle orientation influences the fate that rgc daughters assume after division . to obtain more direct evidence for the proposed lineage changes , we used in utero electroporation ( figures 7a7r ) . for this we electroporated a construct expressing rfp into brains of e14.5 control , knockout , and embryos from r26 males crossed to nescre/ ; r26 females . we used nescre/ ; r26 embryos in order to avoid the observed massive ectopic location of apical and bps . long - term time - lapse experiments during mid - late neurogenesis show that apical progenitors undergo only one division in 24 hr ( noctor et al . , 2004 ) . in order to look at the fate of the daughter cells after one division of apical progenitors , embryos were collected 1 day after electroporation . rfp cells are found in the vz and iz of brains from control , knockout , and knockin embryos ( figures 7b , 7e , 7h , 7k , 7n , and 7q ) . while the electroporated rfp cells have migrated beyond the basal border of the pax6 expression zone in control and knockout animals , the rfp cells are located right at the edge of this expression zone in the minsc - overexpressing animals ( compare figures 7c and 7i with figure 7f ) . to determine the identity of those cells , we used the bp marker tbr2 . in control and mutant brains , tbr2 is expressed in a subset of the rfp - electroporated cells . in control animals , tbr2 is expressed in 23% of the rfp - electroporated cells while this fraction is reduced to about 10% in nescre/ ; minsc embryos . in minsc - overexpressing animals , in contrast , the bp marker is expressed in over 50% of the electroporated cells ( determined as the number of tbr2 , rfp cells divided by the total number of rfp cells , figure 7 t ) . as the percentage of pax6/rfp progenitor cells among all electroporated ( rfp ) cells does not change ( figure 7s ) , these results indicate that a reorientation of the mitotic spindle along the apical - basal axis causes rgcs to preferentially generate intermediate progenitors after division . taken together , our data reveal that spindle orientation along the apical - basal axis is mediated by minsc and is important for promoting neurogenesis . apical - basal divisions are more likely to give rise to intermediate progenitors , and this effect may be responsible for the increased rates of neurogenesis observed upon minsc overexpression . to address the role of nonplanar spindle orientation in cortical development , we have generated a conditional deletion of minsc . unlike drosophila pins , par-3 , par-6 , and apkc , insc has a single , clearly defined mammalian homolog ( katoh , 2003 ; lechler and fuchs , 2005 ; zigman et al . , 2005 ) . in drosophila embryos , insc is exclusively expressed in asymmetrically dividing cells , and no functions other than asymmetric cell division have been reported . mutating centrosomal proteins like asp ( aspm in mice ) ( fish et al . , 2006 , 2008 ) or cnn ( cdk5rap2 in mice ) ( barrera et al . , 2010 ) might affect signaling pathways by disrupting primary cilia and will influence centrosome asymmetry , which was proposed to be important in cortical neurogenesis ( wang et al . , 2009 ) . mutating dynein - binding proteins like lis1 causes defects in spindle morphology and cell migration ( yingling et al . , 2008 ) . therefore , our minsc knockout and minsc - overexpression mice are particularly specific tools to analyze spindle orientation . the spindle orientation defects we observe in minsc - deficient mice are different from the one previously reported for lgn , the mouse homolog of the insc - binding partner pins . in lgn knockouts , the orientation of the mitotic spindle is randomized while lack of minsc causes almost all mitotic spindles to assume a planar orientation . this is in agreement with the functions reported for the two genes in flies and explains why the two genes have different effects on cortical neurogenesis ( konno et al . , 2008 ; our results suggest that intermediate progenitors are more likely to arise from oblique or horizontal divisions ( in which the spindle is oriented oblique or vertical , respectively ) . first , increasing or decreasing minsc expression elevates or reduces the number of neurons , respectively . at the same time , both the total number of apical progenitors and the number of mitotic cells in the vz remain constant . second , minsc levels affect the number of tbr2-positive intermediate progenitors and the number of cells dividing outside the vz . and finally , apical progenitors labeled by electroporation of rfp - expressing plasmids are more likely to give rise to tbr2-positive intermediate progenitors when minsc levels are increased . we propose a model in which minsc influences spindle orientation and thereby regulates the balance between direct and indirect neurogenesis ( figure 8) . whether or not minsc is required for generating all or most bps is not clear . it is remarkable that the terminal forebrain phenotype of minsc mice is similar to the one observed for tbr2 , in which intermediate progenitors are essentially absent ( arnold et al . 2008 ) : in both cases , thickness of the cp is reduced by about 40% . while the outer layers are more affected in tbr2 mice , this could be explained if intermediate progenitors initially form through a spindle orientation - dependent mechanism , but later neurogenesis can also proceed through a partially redundant , minsc - independent mechanism . how could asymmetry be established during progenitor divisions ? quite likely , minsc - independent direct neurogenesis and minsc - dependent indirect neurogenesis might use different mechanisms . both the narrow apical plasma membrane domain and the basal process that connects progenitors to the pial surface should be inherited by only one daughter cell during oblique or vertical division . as bps do not maintain their connection to the apical surface ( gtz and huttner , 2005 ; miyata et al . , 2004 ) but do contain a basal process , the asymmetric inheritance of those structures could contribute to intermediate progenitor formation . for example , intermediate progenitors could simply move out of the vz after s phase because they are not attached to the apical surface . alternatively , apically localized proteins could perform a more direct signaling role . it has been proposed that the asymmetric inheritance of par3 can activate notch signaling in one daughter cell of an apical progenitor ( bultje et al . , 2009 ) . as levels of notch signaling are lower in intermediate progenitors and decreasing levels of notch signaling promotes the formation of intermediate progenitors ( mizutani et al . , one could imagine that the loss of par3 during an oblique division establishes bp fate in one of the two daughter cells . alternatively , the basal process could carry certain signaling molecules , whose asymmetric inheritance alters daughter cell fate ( schwamborn et al . , 2009 ) . how could minsc act on a molecular level ? in drosophila , the expression of insc recruits pins to the apical cortex and acts as a molecular switch for spindle orientation . in the mouse cortex , however , progenitor cells seem to express equal levels of minsc regardless of division orientation . it has been demonstrated that horizontal spindle orientation in epithelial cells depends on apkc - mediated phosphorylation of lgn ( hao et al . , , minsc could simply inhibit this pathway by binding to the apkc / par-3/par6 complex and thereby promote nonplanar orientation of the mitotic spindle . in this model , the role of minsc would not be to instruct apical - basal orientation in a binary manner but to introduce imprecision and cause a degree of stochasticity in the orientation of progenitor divisions . this would explain why minsc expression levels do not decide on the orientation of individual progenitor divisions , but overall changes of minsc expression have a strong influence on the fraction of cells that divide in a nonplanar fashion . it has been proposed that changes in spindle orientation have influenced cortical evolution ( bond et al . , 2002 ; fish et al . , 2008 ; zhang , 2003 ) . the gene aspm , which is required for correct orientation of early proliferative neuroepithelial divisions ( fish et al . , 2008 ) , has evolved adaptively in primates suggesting a functional alteration during primate evolution . aspm is localized at spindle poles and is particularly important for correct planar orientation of symmetric progenitor divisions ( fish et al . , 2008 ) . it has been proposed that adaptations in aspm function have increased the fidelity and number of early symmetric divisions , thereby increasing progenitor pools , neuron number , and brain size ( ponting and jackson , 2005 ) . given that minsc regulates spindle orientation , it could have a similar role in primate evolution . in fact , intermediate progenitors play an important role in cortical evolution : in primates these cells can generate many more than two neurons , thus amplifying the total number of neurons arising from one ventricular progenitor . human and ferret brains contain a population of outer subventricular zone ( osvz ) progenitors that have been attributed a key role in amplifying neuron numbers ( fietz et al . live - imaging experiments have suggested that spindle orientation is crucial for establishing this cell population ( shitamukai et al . , 2011 ; wang et al . , 2011 ) . given that minsc is a key regulator of intermediate progenitor formation , it could regulate osvz progenitor formation as well . in this case , characterization of evolutionary changes in the minsc locus and a functional analysis in the human brain might yield important information on how this unique cell population has arisen in evolution . primary antibodies used were : rabbit anti - minsc ( 1:100 ; zigman et al . ) ; mouse anti--gal ( promega ) ; chicken anti - gfp ( 1:500 ; abcam ) ; rabbit anti - satb2 ( 1:500 ; abcam ) ; rabbit anti - foxp1 ( 1:500 ; abcam ) ; rabbit anti - foxp2 ( 1:500 ; abcam ) ; mouse anti - tuj1 ( 1:500 ; sigma - aldrich ) ; rabbit anti - nestin ( 1:500 ; becton dickinson ) ; rabbit anti - pax6 ( 1:300 ; covance ) ; rabbit anti - tbr1 ( 1:500 ; abcam ) ; mouse anti - map2 ( 1:500 ; chemicon ) ; rabbit anti - tbr2 ( 1:500 ; abcam ) ; rabbit anti - ph3 ( 1:500 ; upstate ) ; and mouse anti - ph3 ( 1:500 ; cell signaling ) . secondary antibodies were conjugates of alexa fluor 488 , alexa fluor 568 , and alexa fluor 647 ( 1:500 ; invitrogen ) . fifteen micron coronal sections of e11.5 and e14.5 embryonic brains paraffin embedded were stained with mouse anti-tub ( 1:1000 ; sigma - aldrich ) , mouse anti-tub ( 1:1000 ; sigma - aldrich ) , and rabbit anti - ph3 ( upstate ) , using the staining protocol described in the supplemental experimental procedures . z stacks with an interval of 0.5 m were taken using a zeiss axiovert 200 m confocal microscope . after 3d reconstruction of the confocal stacks of a dividing cell with the imaris software , five points were placed arbitrarily at different positions of the 3d - rendered plane ventricular surface , and two points were placed at the positions of the two centrosomes . the coordinates of the five points were used to determine the best - fitting plane by orthogonal distance regression . the angle between the vector connecting the two points marking the centrosomes and the normal vector of the regression plane was calculated , and 90 minus the angle was used as the division angle . all calculations were done using the r programming environment . in order to have an estimate of the upper limit of error for the division angle calculation , each of the five points was in turn left out for determining the best - fitting plane . thereby , five planes determined by just four points were received , and the angles for these were determined as well as the standard deviation ( sd ) of the angles .

cells comprising a tissue migrate as part of a collective . how collective processes are coordinated over large multi - cellular assemblies has remained unclear , however , because mechanical stresses exerted at cell - cell junctions have not been accessible experimentally . we report here maps of these stresses within and between cells comprising a monolayer . within the cell sheet there arise unanticipated fluctuations of mechanical stress that are severe , emerge spontaneously , and ripple across the monolayer . this stress landscape becomes increasingly rugged , sluggish , and cooperative with increasing system density . within that landscape , local cellular migrations follow local orientations of maximal principal stress . migrations of both endothelial and epithelial monolayers conform to this behavior , as do breast cancer cell lines before but not after the epithelial - mesenchymal transition . collective migration in these diverse systems is seen to be governed by a simple but unifying physiological principle : neighboring cells join forces to transmit appreciable normal stress across the cell - cell junction , but migrate along orientations of minimal intercellular shear stress .

historically , bipolar disorder has been conceptualized as a disease involving episodic rather than chronic dysfunction . however , increasing evidence indicates that bipolar disorder is associated with substantial inter - episode psychosocial and vocational impairment . here we review the contributions of neurocognitive deficits and structural and functional neuroanatomic alterations to the observed functional impairments . in particular , compelling evidence now suggests that neurocognitive impairments , particularly in the areas of attention , processing speed , and memory , are associated with functional outcome . although investigation of the neural correlates of functional disability in bipolar disorder is only in its nascent stages , preliminary evidence suggests that white matter abnormalities may be predictive of poor outcome . a better understanding of the relationship between neurocognitive and neuroimaging assays and functional outcome has the potential to improve current treatment options and provide targets for new treatment strategies in bipolar disorder .

there is growing recognition that bipolar affective disorder is associated with neurocognitive deficits and allied neuroanatomic and neurophysiologic anomalies [ 1 , 2 ] . while mood states and psychotropic medication usage influence cognition and brain physiology , observations that many of these neurocognitive and structural and functional neuroanatomic abnormalities predate symptom onset or persist during symptom remission raise the possibility that some changes in brain structure and function are central to the pathophysiology of bipolar affective disorder [ 3 , 4 ] . furthermore , accumulating evidence suggests that neurocognitive and neuroimaging factors are associated with clinical outcome , psychosocial functioning , and vocational disability in bipolar disorder . the goal of this review is to synthesize these recent findings to assess their clinical relevance and highlight areas in need of additional research . almost two thirds of bipolar patients have some level of inter - episode functional impairment [ 6 , 7 ] , and about half are unemployed or have reduced occupational functioning . thus , although many individuals with bipolar disorder respond well to treatments designed to reduce affective and psychotic symptoms , the ability to achieve functional recovery the capacity to study , work , engage in recreation , live independently , and engage in romantic relationships may be significantly hindered . therefore , recovery from mood episodes and/or residual symptoms does not necessarily translate into functional recovery . indeed , many patients with bipolar illness do not regain premorbid levels of psychosocial functioning . studies of the lifetime course of bipolar disorder consistently find that during periods of symptomatic recovery , bipolar individuals often continue to experience residual mood symptoms that likely contribute to increased levels of disability and decreased psychosocial functioning . clinical factors in particular disease chronicity , severity , and subsyndromal depressive symptoms are associated with poor functional outcome [ 5 , 11 ] . in addition , as reviewed in detail below , there is growing evidence for a relationship between neurocognitive function , underlying neuroanatomic abnormalities , and functional outcome across a range of severe neuropsychiatric disorders , including bipolar illness [ 1214 ] . a better understanding of the relationship between neurocognitive and neuroimaging measures and clinical outcome has the potential to improve current treatment options and provide targets for new treatment strategies . although there is clear evidence that patients with bipolar disorder exhibit widespread neurocognitive dysfunction during acute episodes of mania and depression , the discovery that these deficits endure during symptom remission raises the possibility that cognitive impairment may represent a trait rather than a state variable . euthymic bipolar patients exhibit limitations in several cognitive domains , particularly in measures of executive function , declarative memory , and sustained attention [ 1 , 1517 ] . although euthymic patients often present with residual affective symptoms that may adversely affect performance on cognitive tests , even those who have been euthymic for months prior to assessment have marked neuropsychological impairments . indeed , several recent meta - analyses of neuropsychological performance in euthymic bipolar disorder have documented impairment across a wide variety of cognitive domains [ 1 , 1517 ] . in the most recent of these meta - analyses , kurtz and gerraty found that nonsymptomatic bipolar patients performed 0.40.9 sd below healthy individuals on measures of attention ( e.g , continuous performance test ; cohen s d = 0.69 ) , processing speed ( e.g , digit symbol substitution ; d = 0.66 ) , working memory ( e.g , digit span backward ; d = 0.65 ) , declarative memory ( e.g , rey or california verbal learning test ; d = 0.81 ) , nonverbal declarative memory ( e.g , visual reproduction subtest from the wechsler memory scale ; d = 0.91 ) , and executive functioning ( e.g , trail making test b ; d = 0.72 ) . observations of neuropsychological deficits in nonsymptomatic bipolar patients suggest that these impairments may be related to the pathophysiology of the illness and , as described subsequently , are also reasonable predictors of psychosocial functioning and disability [ 20 ] . evidence that clinically unaffected relatives of patients with bipolar disorder have subtle neurocognitive impairments suggests that such deficits may reflect genetic liability for the illness . a recent meta - analysis reported small but statistically significant differences ( e.g , d < 0.5 ) for unaffected first - degree relatives compared with healthy individuals in the domains of executive functioning and verbal memory . another recent meta - analysis of 17 published studies included 443 first - degree relatives of bipolar patients and reported cognitive impairments in the range of small to medium effect sizes in the domains of attention ( 0.080.51 ) , verbal learning ( 0.270.33 ) , and executive functioning ( 0.220.36 ) . glahn and colleagues [ 22 ] recently reported that three cognitive tests ( digit symbol coding , object delayed response task , and immediate facial memory ) are genetically correlated with liability for bipolar disorder , suggesting that the same genetic factors that increase the risk of bipolar disorder influence performance on these tests . although many of the neurocognitive impairments found in individuals with bipolar disorder are present during euthymia , it is quite likely that clinical course influences test performance in bipolar disorder . indeed , age at onset , total number of mood episodes , number of manic episodes , number of depressive episodes , and number and duration of hospitalizations are all factors associated with the degree of neurocognitive impairment among individuals with bipolar disorder . furthermore , the use of psychotropic medication could impact neurocognitive functioning in bipolar patients . a recent meta - analysis examining the effects of lithium on cognitive performance in bipolar disorder in 12 studies involving 276 lithium - treated and 263 lithium - free patients found a small but significant impairment in lithium - treated patients in immediate verbal learning and memory ( effect size , 0.24 ) and creativity ( effect size , 0.33 ) . in contrast , no significant impairments were found for delayed verbal memory , visual memory , attention , executive functioning , processing speed , or psychomotor performance . some antidepressant medications have been shown to yield adverse cognitive effects , particularly those with anticholinergic properties . however , other studies have failed to find significant effects of such medications on cognition [ 24 , 25 ] . although few studies have examined neurocognitive performance in unmedicated bipolar patients , goswami and colleagues found no significant differences in neurocognitive test performance between 22 drug - free euthymic bipolar patients and 22 medicated euthymic patients . together , these data suggest that although clinical course and medication usage may influence cognitive performance in bipolar disorder , these effects seem to explain only a fraction of the observed impairments . growing evidence indicates that the neurocognitive impairments contribute to poorer psychosocial functioning and increased functional disability in bipolar disorder [ 1214 , 20 , 27 , 28 ] . for example , executive dysfunction at initial assessment has been linked to lower levels of functional recovery in this population both cross - sectionally and longitudinally [ 13 , 14 , 28 ] . bonnin and colleagues [ 29 ] recently found that subthreshold depressive symptoms and working memory function ( digits backward ) were specific predictors of occupational functioning 4 years later . similarly , bipolar patients with declarative memory deficits are less likely to return to premorbid psychosocial or occupational functioning [ 11 , 14 , 28 , 29 ] . schizophrenia research has recently begun to distinguish between functional capacity ( i.e , an estimate of one s ability to perform tasks relevant to everyday functioning ) and actual performance ( i.e , what one actually does in everyday settings ) . this distinction is important , as performance can be influenced by functional capacity as well as environmental , motivational , and other factors . bowie and colleagues [ 20 ] recently performed a series of confirmatory path analyses to determine how neurocognitive deficit influences real world functioning and disability in bipolar disorder . a total of 130 community - dwelling individuals with bipolar disorder were assessed with neuropsychological tests , symptom measures , and performance - based social and adaptive functional competence measures in three domains of real world functioning ( community and household activities , work skills , and interpersonal relationships ) . in all models , the relationship between neurocognition and outcome these findings imply that functional disability may persist in patients with bipolar disorder even after symptomatic recovery due to neurocognitive and skill deficits . conversely , even if a patient acquires certain neurocognitive skills but continues to experience mild mood symptomatology , changes in real world behavior might lag or not manifest at all . in summary , significant evidence suggests that many individuals with bipolar disorder have at least some degree of neurocognitive deficit . although these deficits may be influenced by clinical course / severity or psychotropic medication usage , they also seem to be associated with the genetic liability for the illness , suggesting that neurocognitive deficits are an important aspect of the presentation of bipolar disorder . finally , neurocognitive impairments impact real world functioning of patients with bipolar disorder . the most consistently documented neuroanatomic abnormalities in adult patients with bipolar disorder are lateral ventricle enlargement ( + 17% ) and increased rates of deep white matter hyperintensities ( odds ratio , 2.49 ) [ 2 ] . reduced area or volume of the corpus callosum is also a robust finding across studies . additional evidence for white matter involvement in bipolar disorder comes from studies finding alterations in white matter tract organization [ 31 , 32 ] and regional white matter volume reductions . in addition , some recent evidence indicates that white matter abnormalities may be stable , trait - based abnormalities that reflect genetic liability to the illness [ 31 , 34 ] . kieseppa and colleagues found decreased left hemisphere white matter volume in bipolar probands and their nonbipolar co - twins . similarly , mcdonald and colleagues found that the genetic risk of bipolar disorder was associated with white matter reduction in the left frontal and temporoparietal regions , suggesting that left frontotemporal disconnectivity may be a genetically controlled neuroanatomic abnormality associated with bipolar illness . finally , in a large dutch twin sample , van der schot and colleagues [ 35 ] found that reduction in overall white matter volume was related to the genetic risk of bipolar disorder , whereas significant environmental correlations were observed for cortical gray matter . in general , findings of gray matter alterations in bipolar disorder are more variable across studies , which is likely at least partially attributable to the now well - documented effects of lithium on gray matter volume [ 2 , 36 ] . although a recent meta - analysis revealed an effect size of 1.17 for reduced volume of the left anterior cingulate in bipolar patients relative to controls , this finding was not significant across studies due to the high between - study heterogeneity [ 2 ] . kempton and colleagues , using voxel - based morphometry in patients with bipolar i disorder , their relatives with major depression , healthy relatives , and controls , found group differences in the left insula , cerebellum , and substantia nigra ; increased left insula volume in particular was associated with genetic preposition to bipolar disorder independent of clinical phenotype . in contrast , increased left substantia nigra volume was specific to those with the clinical phenotype of bipolar i disorder . changes uniquely associated with the absence of a clinical diagnosis in bipolar relatives were observed in the left cerebellum , suggesting that there may be dissociable genetic and phenotypic influences on brain structure in bipolar disorder . given the substantial body of literature on neuroanatomic changes in bipolar disorder [ 2 ] , surprisingly few studies have examined their relationship to outcome . in patients with schizophrenia and those at risk of the illness , this relationship has been examined in several studies . for example , accelerated ventricular enlargement in the 3 years following illness onset was associated with poor outcome in first - episode schizophrenia , whereas a progressive decrement in frontal lobe white matter was associated with greater negative symptom severity . more recently karlsgodt and colleagues found that the structural integrity of medial temporal white matter tracts was predictive of functional outcome in adolescents at high risk of developing psychosis . given the accumulating evidence that disrupted white matter integrity may be central to the pathophysiology of bipolar disorder , white matter abnormalities represent a promising candidate for a neuroanatomic predictor of outcome . two prior studies examined white matter hyperintensities as indicators of treatment resistance and poor outcome in bipolar disorder . moore and colleagues categorized patients with bipolar disorder as good or poor outcome based on treatment response and level of functioning , finding that poor outcome group members had significantly more deep and more severe subcortical punctate white matter hyperintensities relative to those in the good outcome group and healthy controls . regenold and colleagues found that an index of treatment resistance correlated significantly with deep white matter hyperintensity volume , as well as measures of abnormal brain glucose metabolism ( sorbitol and fructose ) in bipolar patients but not in other patients ( i.e , those with schizophrenia and neurologic controls ) . in summary , there is strong evidence to date implicating neurocognitive factors in bipolar patients as key determinants of functional outcome . given that these cognitive abnormalities likely are reflective of underlying abnormalities in brain structure and function , we propose that microstructural white matter alterations may contribute to poor outcome . similar to the literature relating abnormalities of brain structure to poor outcome in schizophrenia , recent functional mri studies also have begun to document such a relationship . in particular , fusar - poli and colleagues found that in youth with prodromal signs of psychosis , clinical and functional improvement over follow - up was associated with a longitudinal increase in activation in the anterior cingulate and right parahippocampal gyrus during performance on an n - back task . similarly , in individuals at high clinical risk of psychosis , sabb and colleagues found that baseline neural activity in the left inferior frontal gyrus during performance on a language processing task was predictive of severity of positive formal thought disorder and poor social outcome at follow - up . to date , no such studies have been conducted in patients with bipolar disorder . however , emerging literature suggests that the neurophysiology of bipolar disorder involves frontal hypoactivation with concomitant disinhibition ( i.e , relative hyperactivation ) of limbic structures [ 44 , 45 ] . the subgenual prefrontal cortex modulates the affective output of limbic and paralimbic structures and cognitive output from the prefrontal cortex . recent evidence from functional mri studies using affective processing paradigms ( ie , reacting to stimuli with positive or negative emotional valence ) suggests that patients with bipolar disorder may disproportionately engage limbic structures during emotionally valent tasks , regardless of mood state [ 46 , 47 ] , although the direction of effects has not been consistent across studies . taken together , these studies suggest that corticolimbic dysregulation may underlie the emotional dysregulation and cognitive impairments associated with bipolar disorder [ 45 , 48 ] . moreover , exaggerated medial prefrontal cortical and subcortical ( putamen and amygdala ) responses to emotional signals have been observed in bipolar i patients and their nonbipolar relatives , suggesting that such responses may represent heritable neurobiological abnormalities underlying bipolar disorder . given that similar cognitive impairments and associated psychosocial and functional disabilities are seen in patients with bipolar disorder and those with schizophrenia , the adaptation of treatment strategies that have proven beneficial for schizophrenia patients may also be efficacious for bipolar patients . in particular , cognitive remediation has been associated with significant , although modest , improvements in cognitive performance and psychosocial functioning in schizophrenia patients . the development of treatments that target cognitive impairments and functional status is an important area of future investigation in bipolar disorder . two small studies have found that rehabilitative interventions such as cognitive remediation and supported employment may be effective in improving vocational outcomes for bipolar patients , but this will require replication in larger investigations . although bipolar disorder historically has been viewed as a disease involving only episodic dysfunction , increasing evidence indicates that this is not the case for a substantial proportion of patients . collectively , the data reviewed above provide compelling evidence that cognitive impairments are present across multiple domains particularly in the areas of attention , processing speed , and memory in most bipolar patients . these difficulties are observable at or soon after illness onset , and persist throughout the course of illness . given that clinically unaffected relatives of patients with bipolar disorder have similar but milder neurocognitive impairments , such deficits may reflect genetic liability for the illness . although investigation of the neural correlates of functional disability in bipolar disorder is only in its nascent stages , given strong commonalities with schizophrenia , it is anticipated that similar relationships between structural and functional neuroanatomic abnormalities and outcome in bipolar patients will be identified . the rich literature in schizophrenia and in individuals at risk of the illness could serve to inform areas of future research needed in bipolar disorder . as recommended by harvey and colleagues , approaches used in schizophrenia research in particular , longitudinal assessment of cognition , neurophysiology , and psychosocial function across variations in clinical state , and separate assessment of functional capacity and real world functioning will be highly informative when applied to the study of patients with bipolar illness . a clear need exists for objective methods of assessing real world functional abilities , as subjective self - assessments are likely to be influenced by current symptoms or other illness features . thus far , few studies have investigated specific aspects of functional status in bipolar patients and their associations with neuroanatomic , clinical , and treatment - related factors . such investigations are critical for understanding the array of determinants of disability in bipolar illness .

the bcl-2 homolog bcl - xl , one of the two protein products of bcl2l1 , has originally been characterized for its prominent prosurvival functions . similar to bcl-2 , bcl - xl binds to its multidomain proapoptotic counterparts bax and bak , hence preventing the formation of lethal pores in the mitochondrial outer membrane , as well as to multiple bh3-only proteins , thus interrupting apical proapoptotic signals . in addition , bcl - xl has been suggested to exert cytoprotective functions by sequestering a cytosolic pool of the pro - apoptotic transcription factor p53 and by binding to the voltage - dependent anion channel 1 ( vdac1 ) , thereby inhibiting the so - called mitochondrial permeability transition ( mpt ) . thus , bcl - xl appears to play a prominent role in the regulation of multiple distinct types of cell death , including apoptosis and regulated necrosis . more recently , great attention has been given to the cell death - unrelated functions of bcl-2-like proteins . in particular , bcl - xl has been shown to modulate a number of pathophysiological processes , including but not limited to mitochondrial atp synthesis , protein acetylation , autophagy and mitosis . in this short review article , we will discuss the functions of bcl - xl at the interface between cell death and metabolism .

according to current models , cell death most often proceeds via either of two relatively independent subroutines , apoptosis , and necrosis [ 1 , 2 ] . for a long time , apoptotic and necrotic instances of cell death have exclusively been identified based on morphological criteria . in addition , while apoptosis was believed to constitute the sole regulated ( i.e. , genetically encoded , and hence susceptible to pharmacological modulation ) modality of cell death , necrosis was viewed as a purely accidental process . recently , a functional classification of cell death mechanisms , based on measurable biochemical features , has been proposed , and the concept of regulated necrosis has gained large consensus . in this scenario , the true relevance of additional processes that were previously catalogued as bona fide cell death subroutines is being reevaluated . in particular , while macroautophagy ( hereafter referred to as autophagy ) turned out to constitute a prominent homeostatic and cytoprotective mechanism [ 5 , 6 ] , autophagic cell death ( a lethal subroutine that is mediated , rather than merely accompanied , by autophagy ) has been shown to occur in a limited number of , mostly developmental , scenarios [ 1 , 7 ] . along similar lines , mitotic catastrophe , a signaling cascade elicited in mitosis - incompetent cells that was initially viewed as a particular case of apoptosis , has recently been proposed to constitute an oncosuppressive mechanism with multiple functional outcomes , including cell senescence as well as apoptotic and necrotic cell death . apoptotic stimuli can be propagated via two distinct , but not entirely disjointed , molecular cascades : extrinsic apoptosis , transducing lethal signals that originate in the extracellular microenvironment , and intrinsic ( also known as mitochondrial ) apoptosis , responding to perturbations of intracellular homeostasis . extrinsic apoptosis can be initiated either by the ligand - induced activation of plasma membrane death receptors ( e.g. , fas / cd95 , tumor necrosis factor receptor 1 ( tnfr1 ) ) or by so - called dependence receptors ( e.g. , deleted in colorectal carcinoma ( dcc ) ) , when the concentration of their ligands falls below a specific threshold . death receptors promote the activation of caspases ( a class of cysteine proteases that play a central role in multiple instances of apoptosis ) via the formation of a multiprotein complex that includes among other components receptor - interacting protein kinase 1 ( ripk1 ) , fas - associated protein with death domain ( fadd ) , cellular inhibitor of apoptosis proteins ( ciaps ) , and multiple isoforms of cellular flice - inhibitory protein ( c - flip ) . such a death - inducing signaling complex ( disc ) allows for the proximity - induced autoactivation of caspase-8 , in turn catalyzing the proteolytic maturation of caspase-3 , the central effector of most cases of apoptosis . the mechanisms whereby dependence receptors are connected to the execution of apoptosis have only recently begun to emerge and appear to involve caspase-9 , a caspase that was long believed to exclusively regulate mitochondrial apoptosis . intrinsic apoptosis can be triggered by a plethora of perturbations in intracellular homeostasis , including among others independent of the initiating stimulus , the signaling cascades that mediate intrinsic apoptosis as well as the prosurvival signals that are generated alongside ( to facilitate the reestablishment of homeostasis ) are opposed to each other at the level of mitochondria [ 1315 ] . if lethal signals prevail , the majority of mitochondria become permeabilized , an event that de facto seals the cell fate . indeed , upon mitochondrial outer membrane permeabilization ( momp ) , ( i ) the mitochondrial transmembrane potential ( m ) , that is , the electrochemical gradient driving atp synthesis as well as many other mitochondrial functions , is rapidly dissipated ; and ( ii ) cytotoxic proteins that are normally secluded within the mitochondrial intermembrane space ( e.g. , cytochrome c ; apoptosis - inducing factor ( aif ) , endonuclease g ( endog ) ) are released into the cytosol , where they promote the activation of caspases as well as of caspase - independent cell death executioner mechanisms . the former relies on the cytochrome c - elicited , datp- , and apoptotic peptidase activating factor 1 ( apaf1)-dependent assembly of the so - called apoptosome , a molecular platform for the activation of the caspase-9 caspase-3 cascade . the latter involves the caspase - independent endonuclease activity of aif and endog as well as the bioenergetic and redox crisis that ensues m dissipation [ 13 , 14 ] . of note , extrinsic and intrinsic apoptosis are not entirely disjointed . indeed , while in some cell types ( e.g. , lymphocytes ) the caspase-8 caspase-3 cascade is sufficient to mediate death receptor - dependent apoptosis , in others ( e.g. , hepatocytes ) , this process requires the caspase-8-mediated cleavage of the bh3-only protein bid , generating a mitochondrion - permeabilizing fragment ( see below ) [ 16 , 17 ] . given its position at the frontier between cell life and death , it is not surprising that momp constitutes a highly regulated phenomenon . so far , two models have been put forward to explain momp in molecular terms [ 14 , 18 ] . on one hand , momp has been suggested to originate at the mitochondrial outer membrane ( om ) , thanks to the pore - forming activity of multidomain proapoptotic members of the bcl-2 protein family , namely , bax and bak [ 14 , 19 ] . on the other hand , it has been proposed that in response to specific triggers momp would stem from the so - called mitochondrial permeability transition ( mpt ) , an abrupt increase in the permeability to small solutes of the mitochondrial inner membrane ( i m ) . in this latter scenario , a critical role has been ascribed to the permeability transition pore complex ( ptpc ) , a large molecular entity assembled at the junctions between the om and the i m by several proteins , including ( though presumably not limited to ) voltage - dependent anion channels ( vdacs ) , adenine nucleotide translocase ( ants ) , and cyclophilin d ( cypd ) [ 13 , 18 ] . importantly , antiapoptotic multidomain members of the bcl-2 protein family , including bcl-2 itself , bcl - xl , and mcl-1 , not only counteract the pore - forming activity of bax and bak by engaging in direct inhibitory interactions , but also ( i ) intercept upstream proapoptotic signals such as those mediated by bh3 only proteins like bad , bid , bim , and bbcr3 ( best known as p53-upregulated modulator of apoptosis ( puma ) ) [ 20 , 21 ] , ( ii ) bind to , hence regulating , several components of the ptpc , including vdac1 and ant [ 2224 ] , and ( iii ) prevent the generation of proapoptotic cytosolic ca waves , either by interacting with inositol 1,4,5-trisphosphate ( ip3)-gated ca channels on the endoplasmic reticulum ( er ) or by limiting the capacity of er ca stores [ 2527 ] . in addition , both pro- and antiapoptotic bcl-2-like proteins have recently been shown to modulate multiple processes that are not directly connected to the execution of cell death , including among others bioenergetic metabolism , mitochondrial functions , mitosis , and autophagy [ 28 , 29 ] . here , we discuss the multifaceted role of bcl - xl , a prototypic antiapoptotic member of the bcl-2 family , at the hub between cell death and metabolism . in humans , bcl - xl is encoded by bcl2l1 , a bcl2-related gene mapping to chromosome 20q11.21 . bcl2l1 was shown from the beginning to code for two distinct protein products , owing to the alternative splicing of bcl2l1 mrna : a cytoprotective factor of 233 residues ( bcl - xl ) and a smaller polypeptide ( 170 residues ) that exerts bcl - xl - antagonizing functions ( bcl - xs ) . similar to bcl-2 , bcl - xl contains four distinct bcl-2 homology ( bh ) domains ( bh1bh4 ) as well as a transmembrane region , through which it localizes at least in part to several membranous compartments , including the om , the er , and the nuclear envelope [ 3032 ] the same does not apply to bcl - xs , which lacks both the bh1 and bh2 domains [ 30 , 33 ] . of note , in most physiological settings the bcl - xl - coding mrna is expressed to higher levels than its bcl - xs - coding counterpart . conversely , bcl - xs often predominates in situations of developmental and pharmacological cell death [ 35 , 36 ] . importantly , a caspase - generated cleavage product of bcl - xl ( lacking an n - terminal fragment ) has recently been shown to mediate neuronal cell death in rodent models of ischemic brain injury , suggesting that chemical inhibitors of bcl-2-like proteins might also be employed ( at least in selected circumstances ) as cytoprotective agents . bcl-2 and bcl - xl were soon recognized as critical antiapoptotic factors , although this function was initially attributed to their ability to mediate antioxidant effects [ 38 , 39 ] . this notion has quickly been abandoned in favor of the so - called rheostat model , proposing that bcl-2 and bcl - xl would physically sequester their proapoptotic counterparts bax and bak in inhibitory interactions [ 4042 ] . in the following decade , along with the discovery of several other members of the bcl-2 protein family , this model has been progressively refined to include the concepts of activating and derepressing bh3-only proteins . according to current viewpoints , the former would promote momp by engaging in direct activatory liaisons with bax and bak , while the latter would do so by competitively displacing bax and bak from inhibitory interactions with bcl-2 , bcl - xl , and mcl-1 . of note , the core concept of the rheostat model as first theorized by stanley korsmeyer in 1993 , that is , that cell death is governed by the balance between pro- and antiapoptotic bcl-2 family members , has remained remarkably unmodified since its original formulation . nevertheless , during the last two decades , antiapoptotic bcl-2 family members , including bcl - xl , have been shown to exert cytoprotective functions via a myriad of mechanisms that do not necessarily rely on their capacity to block the pore - forming activity of bax and bak although at least in some instances they do involve a bax-/bak - antagonizing effect . similar to bcl-2 , bcl - xl prevents the generation of proapoptotic cytosolic ca waves by reducing capacity of er ca stores , an effect that is antagonized by bax and bak [ 2527 ] . moreover , bcl - xl has been shown to critically regulate the opening status of vdac1 , and hence of the ptpc , thus influencing mpt - dependent apoptotic cell death [ 23 , 24 , 44 ] . of note , while some authors proposed that the mpt would stem from an unselectively open conformation of the ptpc , others concluded that the mpt would originate from the closed state of the pore [ 24 , 44 ] . irrespective of this controversy , which has not yet been fully resolved , recent data have confirmed a critical role for the interaction between vdac1 and bcl - xl in the antiapoptotic properties of the latter . interestingly , many other bcl-2 family members such as bax , bak , bid , and bcl-2 appear to interact with ( and hence modulate the activity of ) ptpc components ( i.e. , vdac1 , vdac2 , and ant ) [ 22 , 23 , 46 , 47 ] , suggesting that the crosstalk between these two systems might constitute a particularly important point of functional regulation . however , the actual relevance of the ptpc for the cellular demise in physiological settings remains matter of debate . indeed , mice lacking one or more of the most critical ptpc components ( including all vdac and ant isoforms known thus far ) [ 4850 ] , with the single exception of ppif animals ( lacking cypd ) [ 5153 ] , fail to exhibit remarkable cell death defects in response to ischemic , traumatic , and pharmacological challenges . hence , it seems that at least in physiological settings the complex crosstalk between bcl-2 family members and the ptpc mainly modulates cell - death unrelated cellular functions and impacts on the cellular demise only via indirect circuitries ( see below ) . one of the most central regulators of apoptosis as triggered by perturbations of intracellular homeostasis such as dna damaging conditions , imbalances in redox homeostasis and oncogenic stress is p53 . besides operating as a stress - responsive transcription factor that regulates the synthesis of both pro- and antiapoptotic proteins , including a large panel of bcl-2 family members ( i.e. , bax , bak , bad , bid , puma , bcl-2 , and bcl - xl ) , p53 can also exert apoptotic functions in a transcription - independent fashion [ 56 , 57 ] . in particular , p53 has been shown to operate similar to bh3-only proteins , that is , to promote momp either by engaging in activatory ( though labile ) interactions with bax or by displacing bax and bak from inhibitory liaisons with bcl-2 and bcl - xl . in this setting , the mitochondrial pools of bcl-2 and bcl - xl constitute the main target for the derepressor activity of p53 . in addition , a cytoplasmic pool of bcl - xl appears to work as a puma - sensitive inhibitor of p53 , de facto operating at the interface between p53 transcriptional and transcription - independent functions . thus , bcl - xl exerts cytoprotective effects not only as it antagonizes its proapoptotic counterparts but also as it counteracts the activity of p53 . in addition , bcl - xl has recently been reported to interact with the mitochondrial phosphatase phosphoglycerate mutase family member 5 ( pgam5 ) , a central effector of regulated necrosis [ 62 , 63 ] . hence , although niture et al . did not address this question in a direct fashion , bcl - xl may soon be discovered to regulate necrotic instances of cell death . the hypothesis that bcl-2 family members , notably bax , bad , bcl-2 , and bcl - xl , would influence bioenergetic and intermediate metabolism began to gain consensus along with the discoveries that ( i ) these proteins interact with components of the ptpcs that , in physiological circumstances , regulate various facets of mitochondrial functions ( e.g. , ant , vdac , and glucokinase ) [ 2224 , 44 , 64 ] , ( ii ) these proteins modulate ca homeostasis at the er [ 2527 ] , and ( iii ) p53 not only operates as a potent proapoptotic factor in response to stress but also exerts an homeostatic control over metabolism . in particular , bcl - xl ( i ) reportedly preserves the physiological conformation of vdac , hence promoting the exchange of metabolites , including adp , across the om ; ( ii ) functionally antagonizes bad , which has been found to exert prominent metabolic functions by regulating a mitochondrial multiprotein complex that involves ( among other enzymes ) glucokinase , protein kinase a , and protein phosphatase 1 ; ( iii ) has been shown to lower the concentrations of ca ions within the er , hence quenching the bioenergetic burst that normally results from the opening of ip3-gated ca channels [ 2527 ] ; and ( iv ) binds to cytoplasmic p53 in steady - state conditions , thus at least theoretically modulating its functions related to bioenergetic and redox metabolism [ 54 , 65 , 66 ] . bcl - xl has also been demonstrated to regulate distinct facets of intermediate metabolism in a direct fashion . in neurons , a pool of bcl - xl localized to the i m appears to physically interact with the subunit of the f1fo atp synthase , hence increasing its enzymatic efficiency , stabilizing the m and consequently maximizing mitochondrial atp generation [ 67 , 68 ] . similar functions have been attributed to a truncated variant of mcl-1 that localizes to the mitochondrial matrix . in addition , the transfection - enforced overexpression of bcl-2 has been associated with increased oxygen consumption and higher rates of mitochondrial respiration [ 70 , 71 ] . taken together , these observations suggest a conserved role for bcl-2 proteins in the regulation of atp synthesis . of note , antiapoptotic members of the bcl-2 family have been suggested to exert prooxidant functions , at least under selected circumstances [ 70 , 71 ] . such a ( slight ) prooxidant state , presumably reflecting the ability of bcl-2 and bcl - xl to stimulate mitochondrial respiration [ 67 , 68 , 70 ] , appears to be linked to the interaction of bcl-2-like proteins with small gtpases of the rac family [ 72 , 73 ] and to exert cytoprotective effects by contributing to the maintenance of baseline energetic homeostasis . recent data have indicated that bcl - xl operates ( in a bax- and bak - independent manner ) to limit the intracellular levels of acetyl - coa . acetyl - coa is not only a critical intermediate of the krebs cycle , but also required for protein acetylation , including n--acetylation , that is , the posttranslational modification that consists in the addition of an acetyl moiety ( provided by acetyl - coa ) to the n - terminus of nascent polypeptides . hence , high expression levels of bcl - xl exert cytoprotective effects along with the establishment of a state characterized by decreased levels of virtually all the metabolites involved in the krebs cycle ( but not of glycolytic substrates ) as well as by reduced extents of n--acetylation . although yi and colleagues ascribed such an antiapoptotic state ( which could be reversed by the exogenous supply of citrate and acetate ) only to the inhibition of n--acetylation , reduced levels of reactive oxygen species ( ros ) , which constitute a normal byproduct of mitochondrial respiration , may equally well underpin ( at least part of ) the cytoprotective effects that originate from bcl - xl metabolic functions . in support to this notion , ( i ) the overactivation of several metabolic circuitries ( including glycogenolysis and glutaminolysis ) and the overgeneration of ros have been linked to both apoptotic and necrotic cell death [ 13 , 76 ] ; and ( ii ) a predominantly glycolytic metabolism , as observed in cancer cells even in the presence of normal oxygen levels ( i.e. , the so - called warburg effect ) , reportedly exerts cytoprotective effects as it increases the amounts of reduced glutathione ( a potent antioxidant ) . of note , the pyruvate kinase m2 , a glycolytic enzyme variant that is known to sustain the warburg effect , has been shown to stimulate the expression of bcl - xl at the transcriptional level . although the cytoprotective transcription factor nf-b may play a role in this setting , the precise molecular mechanisms underlying this phenomenon remain to be identified . in addition , protein acetylation has recently been involved in the regulation of autophagy ( see below ) [ 7982 ] , suggesting that bcl - xl might exert a broad control over multiple cellular functions . autophagy is a catabolic pathway driving the lysosomal degradation of cellular constituents such as portions of the cytoplasm , protein aggregates , and dysfunctional / supernumerary organelles . under physiological conditions , autophagy plays a prominent role in the maintenance of intracellular homeostasis [ 5 , 84 , 85 ] . in addition , the autophagic flux is dramatically upregulated in response to a large panel of stress conditions , including ( but not limited to ) glucose and amino acid deprivation , hypoxia , intracellular pathogens , and cytotoxic xenobiotics [ 83 , 86 ] . although in some , mostly developmental , scenarios an autophagic program de facto mediates cell death [ 1 , 7 ] , stress - elicited autophagy near to invariably exerts prominent cytoprotective functions . in line with this notion , both pharmacological and genetic maneuvers that block autophagy most often exacerbate , rather than limit , cell death as triggered by several distinct stimuli . the stress - elicited upregulation of autophagy is a tightly regulated phenomenon , involving distinct molecular sensors and signal transduction cascades that impinge at various levels on the autophagic machinery . a detailed description of the proteins and factors that are involved in the regulation and execution of autophagy largely exceeds the scope of this paper and can be found elsewhere [ 83 , 84 , 87 , 89 ] . nevertheless , it is important to note that in most ( but not all ) settings autophagy critically relies on a class iii phosphoinositide-3-kinase ( pi3k ) enzymatic activity . in human cells , this function is mediated by phosphatidylinositol 3-kinase , catalytic subunit type 3 ( pik3c3 , best known as hvps34 ) , which operates under the control of a multiprotein complex involving among other interactors the haploinsufficient oncosuppressor beclin 1 [ 91 , 92 ] . importantly , by virtue of a bona fide bh3 domain , beclin 1 can physically interact with antiapoptotic members of the bcl-2 protein family , including bcl-2 itself and bcl - xl [ 9496 ] . by interacting with beclin 1 , bcl-2 and bcl - xl de facto prevent the stress - induced activation of autophagy . in line with this notion , both bh3-only proteins ( e.g. , bad ) and chemical inhibitors of bcl-2-like proteins ( e.g. , abt-737 ) have been shown to activate autophagy as they displace beclin 1 from inhibitory liaisons with bcl-2 and bcl - xl [ 94 , 97 ] . interestingly , some bh3-only proteins like bnip3 have been shown to be critical for the execution of specific autophagic programs , such as the selective removal of damaged mitochondria ( mitophagy ) [ 5 , 98 ] , while their relevance in cell death regulation seems rather limited . the binding of beclin 1 to antiapoptotic bcl-2 family members can also be resolved by the phosphorylation of either binding partner [ 100102 ] . conversely , it seems that bcl-2 and bcl - xl do not affect the steady - state levels of the autophagic flux in a direct fashion . of note , tian et al . have recently suggested that bcl-2/bcl - xl - targeting compounds might activate a beclin 1- and pik3c3-independent autophagic program leading to cell death . however , the authors failed to provide robust data to mechanistically explain their findings . besides a direct autophagy - modulatory function stemming from its interactions with beclin 1 and other bcl-2-like proteins [ 94 , 96 ] , bcl - xl is expected to regulate autophagy via less direct metabolic circuitries , notably as it ( i ) controls the efficiency of mitochondrial atp production [ 67 , 68 ] , ( ii ) influences the exchange of critical bioenergetic metabolites ( e.g. , atp and adp ) by ptpc components [ 24 , 44 ] , ( iii ) reduces the intracellular levels of acetyl - coa , and ( iv ) interacts with the cytoplasmic pool of p53 [ 60 , 65 , 66 ] . hence , the expression levels of bcl - xl might also influence the autophagic flux in steady - state , as opposed to adaptive , conditions . this aspect of the crosstalk between bcl - xl and autophagy warrants further investigation . the implication of bcl-2 family members , including bcl - xl , in cell death - unrelated processes is not limited to the aspects of cell biology discussed above [ 28 , 29 ] . for instance , it has recently been shown that bcl - xl is phosphorylated at multiple serine residues ( including s49 and s62 ) in a cell cycle - dependent fashion [ 104 , 105 ] . the mitotic kinase polo - like kinase 3 ( plk3 ) appears to be responsible for the cell cycle - dependent phosphorylation of bcl - xl at s49 ( starting at the s phase and abruptly falling at the onset of mitosis ) , whereas plk1 and mitogen - activated protein kinase 9 ( mapk9 ) have been suggested to catalyze bcl - xl phosphorylation at s62 in response to dna - damaging agents , hence stabilizing a cell cycle arrest at the g2 checkpoint . hence , similar to bcl-2 [ 106 , 107 ] , bcl - xl plays a role in both physiological cell cycle progression and dna damage - induced cell cycle checkpoints [ 104 , 105 ] . a few years ago , rather unspecific inhibitors of bcl-2-like proteins such as abt-737 and abt-263 have generated an intense wave of enthusiasm and quickly entered clinical trials as part of antineoplastic regimens for the treatment of mostly hematological malignancies . one of the most prominent on - target side effects of abt-737 and abt-263 turned out to be a dose - limiting thrombocytopenia [ 109 , 110 ] , linked to the fact that bcl - xl is critical for the survival of platelets . more recently , bcl - xl has also been involved in the adhesive function of platelets [ 112 , 113 ] . hence , abt-737 and abt-263 appear to impair aggregation not only as they trigger the demise of a consistent fraction of circulating platelets but also as they exert consistent thrombocytopathic effects among residual platelets . the implication of the bcl-2 protein family in mitochondrial dynamics as well as the actual relevance of mitochondrial fission / fusion events in apoptosis have been and still are the subject of a vivid debate [ 114116 ] . while a detailed discussion of this topic largely exceeds the scope of this paper , it is worth noting that bcl- xl has recently been shown to interact with ( and stimulate the gtpase activity of ) dynamin - related protein 1 ( drp1 ) , a central component of the mitochondrial fission machinery . in doing so , bcl - xl appears to alter the mitochondrial function of neurons in a manner that stimulates the formation of synapses . as drp1 also participates in the execution of regulated necrosis , mitochondrial dynamics may constitute yet another point of control of cell death - related and -unrelated processes by bcl - xl . bcl-2 and bcl - xl have been reported to negatively regulate the nlrp1 inflammasome , a supramolecular platform that is required for the full - blown activation of caspase-1and hence the production of interleukin ( il)-1 and il-18in response to proinflammatory stimuli [ 118 , 119 ] . in particular , the flexible loop domain of both bcl-2 and bcl - xl ( which is located between the 1st and 2nd helices of the proteins ) appears to engage in physical interactions with nlrp1 , thereby blocking the capacity of the latter to bind atp and oligomerize [ 118 , 120 ] . besides playing a critical role in innate immunity , inflammasomes are crucial for the translation of immunogenic cell death ( a functionally peculiar form of apoptosis ) into a robust adaptive immune response . it is therefore tempting to speculate , yet remains to be formally proved , that the interaction between antiapoptotic bcl-2 family members and inflammasomes may constitute a promising therapeutic target for enhancing the immunogenicity of ( cancer ) cell death . following an initial wave of interest on the role of bcl-2-like proteins in the regulation of apoptosis , several laboratories have refocused their attention on distinct aspects of the biology of pro- and antiapoptotic members of the bcl-2 family [ 28 , 29 ] . during the last decade , this intense investigational effort has led to the identification of several processes that are modulated by bcl-2 family proteins independent of ( or at least not directly impacting on ) their cell death - regulatory functions [ 28 , 29 ] . as discussed in this paper , bcl - xl has been shown to exert a consistent degree of control on various aspects of bioenergetic metabolism , including mitochondrial atp production , ca fluxes , autophagy , and protein acetylation , as well as on several other cellular and organismal processes such as mitosis , platelet aggregation , and synaptic efficiency ( table 1 ) . hence , similar to other bcl-2 family members , bcl - xl appears to operate at critical hubs to coordinately control multiple cellular functions including the three - step switch between homeostatic metabolisms , adaptive responses to stress , and cell death . in this scenario , it is tempting to speculate , yet remains to be formally demonstrated , that bcl-2-like proteins may have originated as regulators of non - apoptotic functions and only later in evolution may have acquired the capacity of control cell death . similar to what happened ( and is still happening ) for p53 the number of cellular functions that are regulated by bcl-2 family members , including bcl - xl , will grow further .

background : deliberate self - harm ( dsh ) is a major under - recognized epidemic in the low- and middle - income countries . this is a large retrospective study form the emergency department ( ed ) of tertiary care center of south india to describe the clinicodemographic features of dsh cases.materials and methods : this is a retrospective study conducted at ed of christian medical college , vellore , india from january 01 , 2011 to december 31 , 2013 . all cases of dsh were included in the study . the demographic details , mode of dsh and clinical outcome were extracted from the electronic medical record . descriptive statistics are presented . chi - square test was used to compare categorical variables . for all tests , a two - sided p 0.05 was considered statistically significant.results:total of 1228 patients were admitted to ed for dsh during the study period . male and female occurred in equal ratio . more than half of the cases occurred among age group below 30 years . consumption of pesticides ( agricultural chemicals ) was the single most common mode of dsh ( 46% ) , especially among men , followed by medication overdose ( 29.8% ) . consumption of plant poison and tablet overdose was higher among women . overall mortality due to dsh was low ( 1.5% ) in our study.conclusion:dsh is under - recognized major public health problem in low - middle income countries like india . most cases occur among young and productive age group and in equal frequencies among men and women . timely and the appropriate institution of treatment can decrease the morbidity and mortality due to dsh remarkably .

deliberate self - harm ( dsh ) is one of the commonest cause of death in the world with a higher rate noted in the low- and middle - income countries . soman et al . from south india reported that dsh was responsible for 6.6% of all deaths . the average age was 42 years for males and 34 years for females and half of all suicides occurred in the age group of 1544 years . reported that women had frequent dsh attempts and the mean age was 24.5 ( 9 ) years . dsh attempts are one of the commonest reasons for emergency department ( ed ) admissions . self - poisoning was the most common method used for dsh in a study done across eight low- and middle - income countries . suicide attempts were common among those from low socioeconomic status , and the common modes were self - poisoning with organophosphorus compounds and overdosage of tablets . the suicide rate was high among the elderly according to a study done in south india , and the common modes were hanging and organo - phosphorous poisoning . insecticide poisoning and self - immolation were associated with high mortality rate in india while in a south american country hanging and jumping from heights had a bad outcome . a wide variety of poisons like insecticides , pesticides , plant poisons , rodenticides is freely available across the country . there is a wide variation in the method of dsh used across different states of india and varies with the socioeconomic status . this study was done to describe the profile of dsh attempts and outcome of patients presenting to the adult ed of our tertiary care hospital in south india . this is a retrospective study , of patients , presenting with dsh to the adult ed of christian medical college , vellore , which is a 45-bed department in south india with an average of 200 admissions daily . all patients more than 15 years old presenting with a history of alleged dsh from january 01 , 2011 to december 31 , 2013 , were included in the study . a retrospective chart review was performed on all the above said patients using the hospital 's electronic medical database . the following were extracted : demographics , age , sex , date of admission , type of dsh used , discharge status , and duration of admission . the categorical variables were expressed in proportion and chi - square test or fisher exact test was used to compare dichotomous variables . for all tests , this study was approved by the institutional review board , and patient confidentiality was maintained using unique identifiers and by password protected data entry software with restricted users . eighty - seven percent of those were medical / surgical emergencies , and 13% were trauma related cases . the prevalence of patients presenting with dsh was 1.08% ( 1228/1,13,243 ) [ figure 1 ] . there was a slight female predominance ( 51.8% ) which was consistent over the study period of 3 years [ table 1 ] . almost half of the patients ( 48.1% ) were in the age group between 20 and 29 years of age . elderly population ( 60 years ) constituted 3.5% of the study population [ table 1 ] . the month - wise distribution of dsh cases over a 3-year period is shown in figure 2 . the time of dsh admissions were as follows , 37.1% ( 456/1228 ) were at nights ( 21000500 h , the following day ) , 29% ( 356/1228 ) were at afternoons ( 12001700 h ) , 18.9% ( 232/1228 ) at evenings ( 17002100 h ) , and 15% ( 184/1228 ) during morning hours ( 05001700 h ) . patient flow in emergency department with classification of dsh baseline characteristics mode of deliberate self - harm among men and women consumption of pesticide chemicals ( agricultural chemicals ) ( 46% ) was the most common mode of dsh followed by tablet overdosing ( 29.8% ) . consumption of plant poisons ( 7.8% ) , near hanging ( 5.3% ) , corrosive chemical ingestion ( 4.7% ) , and ingestion of rodenticides ( 4.2% ) were the other common modalities of dsh . two wild plants of our locality which were consumed for dsh were oleander ( 52/96 ) and oduvanthalai ( 42/96 ) . the other plants that were used for dsh were henna and eucalyptus with one case each of the leaves being consumed . rare modes of dsh ( 2% ) were hydrocarbon ingestion ( 12 ) , hair dye ingestion ( 5 ) , self - infliction with sharp objects ( 4 ) , ingestion crushed glass pieces ( 2 ) , burns ( 1 ) , and ingestion of copper oxychloride ( 1 ) [ table 2 ] . the method of dsh more preferred by women were plant poisons ( 66.3% vs. 33.7% , p = 0.006 ) , corrosives ( 63.8% vs. 36.2% , p = 0.047 ) and tablet overdose ( 64.9% vs. 34.1% , p = 0.001 ) while agricultural chemicals were more likely to be consumed by men ( 59.5% vs. 40.5% , p = 0.001 ) for dsh . the preferred mode of dsh by the males and females is shown in figure 3 . mode of deliberate self - harm month wise distribution of the number of patients with dsh for 3 years the outcome was studied by the condition at discharge from hospital and the number of days in hospital . majority ( 97.9% ) of the patients were discharged alive from the hospital while 19 ( 1.5% ) died in the hospital . the discharge status of 7 patients was mentioned as either left against medical advice or discharged at request . the length of hospital stay was 2 days among 634 ( 51.6% ) patients , between 3 and 6 days among 374 ( 30.5% ) , between 7 and 13 days among 151 ( 12.3% ) and 2 weeks or more in 69 ( 5.6% ) patients , respectively [ table 3 ] . the objective of this study was to describe the demographic features , mode of dsh and outcome of patients presenting with dsh to a tertiary care hospital in south india . nearly , 1% of our admission in the ed is related to dsh . in our study , the male : female ratio was 0.93 with a slight female predominance ( 51.8% ) , while a study done in a low socioeconomic country reported a male to female suicide ratio of 1.7 . a study conducted in a middle - income country showed that women had frequent dsh attempts than men . a similar study revealed the average age was 42 years for males and 34 years for females with half of all suicides occurring in the age group of 1544 years . the average age commonly resorting to dsh was 24.5 ( 9 ) years as reported by nojomi et al . in our study , dsh was common among the young with 82.7% patients being < 40 years of age . almost half of the patients ( 48.1% ) were in the age group between 20 and 29 years of age . patients 60 years constituted 3.5% of the dsh attempts reported , and dsh was not uncommon among the elderly . dsh attempts usually present to the ed and self - poisonings were the common mode of dsh according to a study done in eight low- and middle - income countries . in this study on an average , at least , one dsh presented to ed every day . self - poisoning was the most common method of dsh , and agricultural chemicals ( 46% ) were commonly used to commit dsh . other studies from india also reported insecticide poisoning and tablet overdosage as the frequent modes of dsh . the other modes of dsh reported were near hanging , self - immolation , jumping from heights , self - infliction , inhalation of automobile gases , etc . the less frequently reported modes of dsh in this study were consumption of plant poisons ( 7.8% ) , near hanging ( 5.3% ) , corrosive chemical ingestion ( 4.7% ) , and ingestion of rat killer poisons ( 4.2% ) . multiple modes of dsh were observed in 3 patients , one patient presented with near hanging after consuming insecticide poison , the second patient consumed a combination of kerosene , insecticide and paint and the third patient consumed zinc phosphide along with paracetamol tablets . since the hospital in which the study was done has a separate triage and resuscitation policy for burns , exact numbers could not be presented . study on suicides in the same geographical area reported a total mortality rate of 82.2/100000 population . as the age group affected was from 15 to 40 years the total years of life lost was also high ( 26.9 years ) . however , in this study , of the total 1228 patients with attempted dsh 1202 ( 97.9% ) patients were discharged alive from the hospital , while 19 ( 1.5% ) died in the hospital . this reflects that though suicide attempts posed a high risk for death , hospitalization and aggressive treatment can positively affect the outcome . on further analysis , it was found that of the adult dsh attempts 51.6% were discharged from hospital by 2 days . the length of hospital stay was between 3 and 6 days for 30.5% of patients , between 7 and 13 days for 12.3% and 2 weeks or more 5.6% of the study population . psychiatric illness such as depression and personality disorders were found in majority of first attempters of dsh . alcohol abuse , ethnicity , low income , marital discord , loss of job , and change in weather conditions were also associated with dsh . all patients with dsh attempt should receive a psychiatric evaluation and follow - up should be arranged with a general practitioner or a psychiatrist . preventive health activities such as psychiatric screening and follow - up , socioeconomic upliftment , counseling regarding difficult times in life such as loss of job , marital discord , and alcohol abuse will result in decreased dsh attempts . banning and restricting the use of fatal agricultural chemicals will reduce the morbidity and mortality associated with dsh in low socioeconomic countries . hazard classification of agricultural chemicals should also include their dsh potential and warnings issued accordingly . since still a large number of dsh attempts are with agricultural chemicals , political will , and legal changes regarding the sale of poisonous chemicals can indirectly lead to a decrease in mortality and morbidity associated with dsh attempts . the retrospective nature of this study did not provide information into the risk factors and co - morbid conditions associated with dsh . the retrospective nature of this study did not provide information into the risk factors and co - morbid conditions associated with dsh .

purposedense , vision - obscuring calcification on the posterior aspect of silicone intraocular lenses ( iols ) is often not amenable to neodymium : yttrium - aluminum - garnet capsulotomy , and , in prior reports , has required iol exchange . we report the successful removal of dense calcium deposition on the posterior surface of a three - piece silicone lens using pars plana vitrectomy ( ppv).materials and methodsa 23-gauge ppv was performed using the stellaris vitrectomy system . a light pipe was used to retroilluminate the iol , and a dense fibrous tissue setting with a low cut - rate and high aspiration rate was able to clear the visual axis of the dystrophic calcification without damaging the iol optic.resultsvisual acuity improved from 20/100 to 20/25.conclusionsmall - gauge ppv may be utilized to remove dense dystrophic calcium deposits on the lens surface in lieu of iol exchange .

dense , vision - obscuring calcification on the posterior aspect of silicone intraocular lenses ( iols ) is often not amenable to neodymium : yttrium - aluminum - garnet capsulotomy , and , in prior reports , has required iol exchange . we report the successful removal of dense calcium deposition on the posterior surface of a three - piece silicone lens using pars plana vitrectomy ( ppv ) . a light pipe was used to retroilluminate the iol , and a dense fibrous tissue setting with a low cut - rate and high aspiration rate was able to clear the visual axis of the dystrophic calcification without damaging the iol optic . small - gauge ppv may be utilized to remove dense dystrophic calcium deposits on the lens surface in lieu of iol exchange . dystrophic calcification of the silicone intraocular lenses ( iols ) is a rare complication that can diminish visual acuity and contrast sensitivity . dystrophic calcification has been reported with the use of silicone iols after vitreous hemorrhage or in patients with asteroid hyalosis , and it has been primarily treated with lens explantation , as neodymium : yttrium - aluminum - garnet ( nd : yag ) laser treatment often can not remove dense deposits.1,2 the first report was published in 2004 by wackernagel et al describing the composition of deposits on an explanted silicone lens 4 years after implantation.3 recently , mamalis et al reported the presence of calcium and phosphate in 16 explanted opacified silicone lenses from various manufacturers ; 86.4% of those lenses were in patients with confirmed asteroid hyalosis . concurrently , the authors analyzed 111 calcified hydrophilic acrylic lenses ; none were associated with a history of asteroid hyalosis.4 previous treatments of lens opacification have relied on the lens explantation alone with or without subsequent reimplantation of a new lens.14 this requires a large incision and risks refractive change . herein , we describe a novel approach to treating patients with vision - obscuring dense calcification of silicone lenses without explantation of the iol . a 78-year - old woman underwent bilateral phacoemulsification and three - piece clariflex silicone iol insertion ( abbott medical optics inc , santa ana , ca , usa ) . she had no significant past medical history and a past ocular history of asteroid hyalosis , greater in the right eye , and wet macular degeneration in the left eye with a central scar . also , 9 years later , she described decreased vision in her right eye . visual acuity was 20/100 in the right eye and count - fingers in the left eye . examination revealed dystrophic calcification on the posterior surface of the right intraocular lens ( figure 1 ) . at that time , a nd : yag capsulotomy was unable to clear the calcification . to access the dystrophic calcification , a pars plana approach was taken using a stellaris pc 23-gauge vitrectomy system ( bausch and lomb incorporated , bridgewater , nj , usa ) . after performing a core and anterior vitrectomy , a dense fibrous tissue setting was used with a high aspiration rate of 600 and a variable cut rate less than 1,500 cuts / minute to slowly remove the calcified material from the posterior aspect of the iol , along with the posterior capsule . after clearing the central optic , her visual acuity was 20/25 without correction on postoperative day one , and the central optic was clear of dystrophic material ( figure 2 ) . at 1 year , no material had reaccumulated on the central optic , and her vision remained stable at 20/25 ( figure 3 ) . calcification of the posterior surface of silicone iols in patients with asteroid hyalosis is a rare but well - described late complication after cataract surgery.2 prior reports in the treatment of this dystrophic calcification have required iol explantation or exchange . a case series studying 22 iol exchanges identified complications in 36.4% of procedures , including corneal decompensation , loss of capsular support , vitreous prolapse , and an uncertain refractive outcome.57 in comparison , the united kingdom national ophthalmology database study of vitreoretinal surgery found only 7.8% of pars plana vitrectomies ( ppv ) had an intraoperative complication , the most common of which were iatrogenic retinal breaks ( 3.2%).8 the use of a high - speed , small - gauge vitrectomy system to clear calcification with lasting results via a posterior approach allows use of the existing iol with a more rapid surgical and visual recovery compared to iol exchange . patients maintain the same intraocular optics achieved immediately after cataract surgery and the chance of refractive alteration associated with iol exchange is minimized . in theory , removing the asteroid bodies during vitrectomy decreases the risk of future calcium deposition on the silicone iol optic . in the interest of saving a patient from the risks and refractive uncertainty of an iol exchange , small - gauge ppv may be utilized to remove dense dystrophic calcium deposits from the lens surface .

so far , a fatty pancreas has been related to obesity and the ageing processes in the body . the current list of pathogenetic factors of the condition is clearly extended with genetically conditioned diseases ( cystic fibrosis , shwachman - diamond syndrome and johanson - blizzard syndrome ) , pancreatitis , especially hereditary and obstructive , metabolic and hormonal disorders ( hypertriglyceridemia , hypercholesterolemia , hyperinsulinemia and hypercortisolemia ) , alcohol overuse , taking some medicines ( especially adrenal cortex hormones ) , disease of the liver and visceral adiposis . as regards lipomatosis of that organ resulting mainly from dyslipidemia and hyperglycemia , the term nonalcoholic fatty pancreas disease was introduced . experimental studies on animals and histological preparations of the pancreatic fragments show that the lipotoxicity of the collected adipocytes collected ion the organ release a cascade of proinflammatory phenomena , and even induces the processes of carcinogenesis . pancreas adiposis is best defined in computed tomography and magnetic resonance imaging . however , a series of works proved the usefulness in the diagnostics of that pathology of transabdominal and endoscopic ultrasonography . in that method , the degree of adiposis was based on the comparison of echogenicity of the pancreas and the liver , renal parenchyma , spleen and/or retroperitoneal adipose . recently , the evaluation was expanded by the evaluation of the degree of pancreatic adipose with the pancreas - to - liver index , utilizing to that end a special computer program . according to our experience , the simplest solution is the method utilized by us . on one crosssection of the body of the pancreas , its echogenicity is assessed in comparison to retroperitoneal adipose and the visibility of the splenic vein , pancreatic duct and the major retroperitoneal vessels . depending on the visualization of these structures , it is possible to determine the degree of pancreas adiposis . such a study applies to 250 people , in whom the adiposis was detected in 16.5% , which is close to other cohort us examinations results .

the presented data from literature and own determinations prove that the adiposis of the pancreas should be treated more seriously , since it is often the symptom of a multi - factor damage to that organ . the identification of these factors is supposed to induce to apply a well - thought out health promoting strategy , which with time may contribute to risk reduction , especially of cardiovascular accident and diabetes . our manner of classification of pancreas adiposis seems simple and possible to be applied with the use of any class of ultrasound device . authors do not report any financial or personal connections with other persons or organizations , which might negatively affect the contents of this publication and/or claim authorship rights to this publication .

objective : this study was carried out to know the level of awareness regarding orthodontic procedures among preadolescents as there is very high prevalence of malocclusion.methods:this cross - sectional study was conducted among a sample of 1010 subjects with a mean age of ( in years ) was 13.02 2.146 . a self - administered structured questionnaire proforma was used . pilot study was done to validate the questionnaire , which was constituted of nine items . the student 's t - test and anova test along with stepwise multiple linear regression were applied for the statistical evaluation of means . the level of significance was set at 0.05.results:the overall awareness of orthodontist among the school going children was 45.1% . the knowledge of orthodontic procedures was significantly higher among girls ( 4.46 1.671 ) when compared to boys ( 4.00 1.489 ) . when the results were compared according to the area of location most of the students in the urban areas gave a positive response regarding awareness when compared to children in the rural community.conclusion:this group of preadolescents showed moderate level of awareness regarding orthodontic procedures as they mentioned that it helps in esthetics , better oral hygiene , mastication , and healthy lifestyle .

malocclusion can be defined as an occlusion in which there is a molar relationship between the arches in any of the planes of spaces or in which there are anomalies in tooth position beyond the normal limits . individuals with malocclusion usually have feeling of shame about their facial appearance and also feel shy in society . various factors such as adverse oral habits , anomalies in number of dentition , shape , and developmental position of teeth can cause malocclusion . , there is a need to identify the awareness levels of children with respect to oral health as children play an important role in filling healthy lifestyle for a lifetime . age groups between 12 and 15 years would be benefitted with the knowledge of orthodontic treatment as orthodontic treatment in early ages could be beneficial in preventing further malocclusion complications . furthermore , it may assist the orthodontist in educating the patients and their parents and in providing advice . orthodontic treatment , more than improving the quality - of - life , can bring physical , psychological , and social changes . the major benefits of orthodontic treatment are to improve the physical function , prevention of tissue damage , and correction of esthetic component . until date , there are very few studies , which evaluate the level of awareness among preadolescents regarding orthodontic treatment so , this study was planned . an epidemiological survey was conducted during the period of month between september and december 2013 . a multi - staged simple random sampling technique was used for selecting six schools ( three in urban areas and three in rural areas ) in bilaspur district . the students in the age group of 12 - 15 years were approached from different classes . pilot study was done to validate the closed - ended questionnaire , which was constituted of nine items as : are you aware of an orthodontist , have you seen irregular teeth , do you believe that straitening the teeth makes better smile , helps in mastication , better oral hygiene , easier to speak , healthy lifestyle , are you aware of the duration for braces treatment , are you aware of the cost of orthodontic treatment . there was single examiner for obtaining details of the questionnaire from the subjects with its intra - examiner reliability that is , = 0.87 . after obtaining the prevalence of awareness regarding orthodontic treatment from the subjects , a total sample was obtained using a sample size calculation formulation taking consideration of prevalence obtained . from all the students pursuing education in each of the selected schools , all the available students between the selected age group and who agreed to participate in the survey were selected , that formed a sample size of 1010 subjects with a mean age of ( in years ) was 13.02 2.146 . a survey proforma was prepared using a self - administered structured questionnaire written in english to assess the level of awareness regarding orthodontic procedures . the selected students in all the schools were invited to participate in the survey during the prescheduled time . the purpose of this study was informed and explained to the students . those who voluntarily agreed to participate in the survey and gave a written informed consent were asked to answer the questionnaire . after scoring data was analyzed using the statistical package for social sciences version 16.0 software . descriptive statistics were obtained and mean percentage scores , standard deviation , and frequency distribution were calculated to know the level of awareness . the student 's t - test and anova test were applied for the statistical evaluation of means . after scoring data was analyzed using the statistical package for social sciences version 16.0 software . descriptive statistics were obtained and mean percentage scores , standard deviation , and frequency distribution were calculated to know the level of awareness . the student 's t - test and anova test were applied for the statistical evaluation of means . the total sample composed of 1010 subjects , including 556 boys and 454 girls ; they were further categorized according to the area of location as 606 belong to urban locality and 404 to remote areas . the age of this study population varies from 12 to 15 years as mentioned in table 1 . age - wise awareness of children toward orthodontic procedures using anova test the overall awareness of orthodontist among the school going children was 45.1% . around 55.0% have answered that they had seen irregularity of dentition . when it was asked that straightening of teeth makes better smile 69.8% replied positively . orthodontic treatment helps in mastication was in view of 55.2% students , 55.3% said that it makes better oral hygiene , 45.0% mentioned that after treatment , we will be easy to pronounce and also results in a healthy lifestyle . only 19.9% were aware of the duration of orthodontic treatment and 35.2% exactly knew the cost of orthodontic procedure . the mean scores of awareness about orthodontic procedures were significantly higher among girls ( 4.46 1.671 ) as compared with boys ( 4.00 1.489 ) as illustrated in table 2 . when the results were compared according to the area of location most of the students in the urban areas gave a positive response regarding awareness as compared to children in the rural community [ table 3 ] . awareness of children toward orthodontic procedures using student 's t test awareness of children toward orthodontic procedures according to location using student 's t test anova test revealed a significant difference according to age wise regarding the awareness of orthodontist in which children of 14 years were more aware ( 4.35 1.499 ) and children of 12 years were less aware ( 3.61 1.203 ) as mentioned in table 1 . stepwise multiple linear regression showed that best predictors in descending order according to awareness of orthodontic procedure are age , gender , and area of residence [ table 4 ] . malocclusion is the second most common of the dental diseases in children and young adults , next to dental caries . the aim of orthodontic procedure is to improve dental occlusion , which results in better smile and good functioning in harmony with the face . overall , it has been seen as an increase in awareness of orthodontic treatments as a dental specialty among children as well as adults . a similar fashion has been observed among nigeria population with an associated increase in orthodontic treatment care . many individuals are aware that orthodontic treatment of malocclusion and craniofacial abnormalities , by ensuring proper relationship of temporomandibular joint , may improve phonation , facial esthetics , with beneficial effects on the general and oral health , results in improvement in the quality - of - life . information about the oral health knowledge among indian population is still very limited , especially for rural people , who constitute more than 70% of the population . this type of study can give an indication of changing attitudes toward malocclusion among preadolescents . around less than half of the participants were aware of an orthodontist and the procedures done by them . in the study done by kaur found the level of dental awareness in parents of preschool children in the indian context which revealed a poor level of dental awareness in those parents . data showed that girls were better aware of treatment of malocclusion than males , which were in agreement with other studies . it might be explained on the basis that girls care more about their appearance and would tend to be more educated about their oral health and irregularity in dentition . however , some studies had shown that boys significantly higher knowledge scores as compared to females . similarly , urban children had shown more positive attitude like visiting a dentist in a study done by singh et al . in 2012 . considering the influence of age on awareness of orthodontics , similar findings were seen in a study conducted by friedman et al . , in 1976 . in our survey , we found a moderate awareness in children toward orthodontic awareness . this group of preadolescents showed moderate level of awareness regarding orthodontic procedures . as they believe that it helps in esthetics , better oral hygiene , mastication , and healthy lifestyle .

parasites are a well - known threat to nonhuman primate ( nhp ) populations , and potentially cause zoonotic diseases in humans . in this study , the basic data was provided of the parasites in nhps and the molecular characterization of the enterocytozoon bieneusi , giardia duodenalis , cryptosporidium spp . , and entamoeba spp . were reviewed , which were found in these samples . a total of 3349 fecal samples were collected from 34 species reared at 17 districts in zoos , farms , free - range , or research laboratories , and examined microscopically . eleven genera of intestinal parasites were detected : five genera of protozoans ( isospora spp . , entamoeba spp . , giardia sp . , cryptosporidium spp . , and cyclospora spp . ) and six genera of helminths ( trichuris spp . , strongyloides spp . , ascaris spp . , physaloptera spp . , ancylostoma spp . , and enterobius spp . ) . the overall sample prevalence of parasitic infection was 54.1% ( 1811/3349 ) . entamoeba spp . was the most prevalent ( 36.4% , 1218/3349 ) . the infection rate was the highest in free - range animals ( 73.0% , 670/918 ) ( p < 0.01 ) and guangxi zhuang autonomous region ( 64.8% , 566/873 ) . mixed infections were mostly detected for entamoeba spp . , trichuris spp . , and strongyloides spp .. molecular characterization was reviewed of enterocytozoon bieneusi , giardia duodenalis , cryptosporidium spp . , and entamoeba spp . , as these are zoonotic species or genotypes . this parasitological data for nhps in china , provides important information for veterinarians and public health authorities for the elimination of such parasites and monitor the potential transmission of zoonotic infections from nhps .

nonhuman primates ( nhps ) , with their high level of genetic homology to humans , make them invaluable experimental models for biomedical research ( messaoudi et al . , 2011 , zhang et al . , 2014 ) . however , they are also an increasingly important source of emerging zoonotic diseases in humans , including human immunodeficiency virus ( hiv ) , ebola virus , malaria , etc ( poinar , 2009 , miller et al . , several intestinal parasites occur in nhps , causing asymptomatic or only mild disorders ( karim et al . potentially zoonotic protozoans ( including enterocytozoon bieneusi , giardia duodenalis , cryptosporidium spp . , and entamoeba spp . ) could be maintained and transmitted with the attendant risk of human outbreaks originating in such animal reservoirs ( legesse and erko , 2004 , ye et al . , 2012 ) . the health of nhps is therefore important not only in terms of management objectives , but also concerning public health . compared with developed countries in america and europe , china has relatively rich primate resources and is currently a leading producer and major supplier of nhps to the international market ( zhang et al . , 2014 ) . nhps are commonly maintained in zoos , natural reserves , and zoological gardens by different feeding habitats in china ( karim et al . therefore , it is important to understand the epidemiology of such intestinal parasites and their potential transmission from nhps to humans . the molecular characterization of nhp parasites is increasingly being studied ( berrilli et al . , 2011 , , 2012 , betson et al . , 2014 , li et al . , 2015a , li et al . , 2015b ) , but there is a lack of comprehensive studies on the intestinal parasites in nhps . here , the prevalence of parasites in nhps in china has been reported and the molecular characterization of the enterocytozoon bieneusi , giardia duodenalis , cryptosporidium spp . , and entamoeba spp . this study was conducted in accordance with the chinese laboratory animal administration act ( 1988 ) . the research protocol was reviewed and approved by the research ethics committee of henan agricultural university . appropriate permission was obtained from the director of animals and properties before the samples were collected . veterinarians were notified of the parasitic infections identified in nhps as soon as possible to expedite their management . a total of 3349 fresh fecal specimens were collected from 17 districts in two cities ( beijing and shanghai ) , one autonomous region ( guangxi zhuang autonomous region ) , and eight provinces ( hebei , henan , hubei , hunan , guangdong , sichuan , yunnan , and shanxi ) in china during the period between july 2009 to april 2015 ( fig . 1 ) . 912 fecal specimens were subsequently collected from animals in zoos , 1402 from farms , 918 from free - range , and 117 from those in research laboratories ( table 1 ) . fresh fecal samples from captive nhps , which were kept in separate pens during the day , were collected in the early morning . the specimens from free - living animals were immediately collected from the ground after defecation . each specimen ( about 10 g ) was collected into a plastic container and labelled with the number , district , species , and clinical symptoms of the animal . specimens were transported to the laboratory as soon as possible and stored in 2.5% ( w / v ) potassium dichromate solution at 4 c prior to microscopy . the fecal specimens were sieved through a sieve ( 7.62 cm diameter ) with a pore size of 245 m , transferred into a 50 ml centrifuge tube containing water , and precipitated by centrifugation at 5000 rpm for 10 min . a portion of each specimen was microscopically examined to detect protozoan and helminthic parasites with both sheather 's sugar flotation technique and lugol 's iodine staining ( huang et al . , 2014 ) . wet smears were examined with a bright - field microscope at 100 and 400 magnification to determine the shape , size , and colour of the eggs / cysts . for giardia duodenalis , a total of 1882 fecal specimens from nhps were examined and characterized by ssrrna ( appelbee et al . , 2003 ) , triosephosphate isomerase ( tpi ) ( sulaiman et al . , 2003a ) , glutamate dehydrogenase ( gdh ) ( cacci et al . , 2008 ) and beta - giardin ( bg ) gene ( cacci et al . , 2002 ) . 2660 specimens were identified for cryptosporidium spp . by pcr amplification of the 18s rrna ( xiao et al . , 2001 ) , 70 kda heat shock protein ( hsp70 ) ( xiao and ryan , 2008 ) and genotyped by 60 kda glycoprotein ( gp60 ) gene ( alves et al . , 2003 ) . for enterocytozoon bieneusi , there were a total of 1882 fecal specimens from nhps that were screened and genotyped by ssu rrna its gene ( sulaiman et al . , 2003b ) ; for entamoeba spp . , 531 specimens from 1059 entamoeba spp . positive samples by microscopy , were randomly selected for pcr amplification based on ssu rrna , using the specific primers of e. histolytica ( clark and diamond , 1991 ) , e. dispar ( clark and diamond , 1991 ) , e. moshkovskii ( ali et al . , 2003 ) , e. nuttulli ( verweij et al . , 2001 ) , e. coli ( tachibana et al . , 2009 ) and e. chattoni ( tachibana et al . , 2009 ) in order to identify the molecular characterization . the infection rates were compared with a test , and differences were considered significant at p < 0.01 . eleven genera of intestinal parasites ( five protozoan and six helminths genera ) were found in the nhps ( fig . 2 ) . were the most frequently detected species , with an incidence of 36.4% ( 1218/3349 ) , followed by trichuris spp . the ratio of intestinal parasitic infections ranged from 46.0% to 73.0% among the four feeding habitats ( zoos , farms , free - range , and research laboratories ) ( table 1 ) . the highest infection rate was found in those animals that were the free - range ( 73.0% , 670/918 ) , followed by those in research laboratories ( 63.2% , 74/117 ) , with lower infection rates at zoos ( 46.3% , 422/912 ) and farms ( 46.0% , 645/1402 ) ( p < 0.01 ) . the sample prevalence of infection ranged from 32.6% to 64.8% among the 11 sampling locations . the guangxi zhuang autonomous region had the highest rate ( 64.8% , 566/873 ) , and the lowest was found in guangdong province ( 32.6% , 107/328 ) ( table 2 ) . the majority ( 74.3% , 1345/1811 ) of infected nhps carried one parasitic species , 22.2% ( 402/1811 ) carried two parasitic species , and only 3.5% ( 64/1811 ) carried three or more parasite species ( table 2 ) . the parasites most often involved in mixed infections were entamoeba spp . , trichuris spp . , and strongyloides spp . six families , 20 genera , 34 species of nhps , and 3349 individual specimens were detected , and the infections rates ranged from 0% to 100% in different nhp species ( table 1s ) . 6.5% ( 122/1882 ) of specimens tested for giardia duodenalis were positive by pcr analysis . the assemblages a ( n = 4 ) and b ( n = 118 ) were found , both which have zoonotic potential ( table 3 ) . thirty - two assemblage b isolates with data at all three loci yielded 15 multi - locus genotypes ( mlgs ) ( including 2 known and 13 new ) ( karim et al . the occurrence of giardia duodenalis assemblages in different species of nonhuman primate species are shown in table 2s . for cryptosporidium spp . , 0.7% ( 19/2660 ) were positive by pcr amplification ( karim et al . 73.7% ( 14/19 ) of the positive specimens were found to be cryptosporidium hominis , whilst 26.3% ( 5/19 ) were c. muris . the subtypes of the c. hominis were identified as iba12g3 ( 7/14 ) and iia17 ( 1/14 ) by gp60 gene sequence analysis ( table 4 ) . the occurrence of cryptosporidium spp . and subtypes in nonhuman primate species based on pcr analysis are shown in table 3s . for enterocytozoon bieneusi , there were 16.3% ( 306/1882 ) positive specimens detected by pcr analysis . altogether , 34 its genotypes were observed , including 16 known genotypes ( type iv , d , o , henan v , henan - iv , peru8 , pigebits5 , pigebits7 , ebpa , ebpc , ebpd , peru11 , beb4 , beb6 , i , and cs-1 ) and 18 new genotypes ( cm1 to cm18 ) ( table 5 ) . the new genotypes cm1 to cm3 , cm6 , cm 8 , cm 10 to cm 17 belong to the previously described group 1 , which have zoonotic potential . genotypes cm5 , cm7 , and cm9 clustered with group 2 , whereas genotypes cm4 and cm18 formed new cluster ( karim et al . , 2014b , karim et al . the occurrence of enterocytozoon bieneusi and genotypes in different species of nonhuman primate species are shown in table 4s . for entamoeba spp . , the overall amplification efficiency was 87.19% ( 463 ) among the 531 positive specimens but only entamoeba dispar ( 72.69% , 386/531 ) and entamoeba coli ( 54.05% , 287/531 ) were amplified successfully . the mixed infections with e. dispar and e. coli were 27.1% ( 144/531 ) ( unpublished data ) . this study demonstrates a high sample prevalence ( 54.1% , 1811/3349 ) and diversity ( five protozoan genera and six helminths genera ) of intestinal parasites in nhps in china . similar infection ratio was found in pet macaques ( 59.1% , 52/88 ) in indonesia ( jones - engel et al . , 2004 ) , a zoo in malaysia ( 54.5% , 54/99 ) ( lim et al . , 2008 ) , and pet monkeys in cameroon ( 51.1% , 24/47 ) ( pourrut et al . , 2011 ) . a diversity of intestinal parasites is frequently reported to infect nhps ( jones - engel et al . , 2004 , , 2005 , lim et al . , 2008 , pourrut et al . , 2011 ) . greater parasite species diversity was observed in ta national park , cte divoire ( with nine protozoans and 14 helminths in 3142 specimens ) ( kouassi et al . , 2015 ) . several studies had reported entamoeba spp . as the most prevalent intestinal parasites in nhps ( pourrut et al . , 2011 ) , whereas others reported that strongyloides spp . were the most prevalent ( gillespie et al . , 2005 ) . all five genera of protozoans detected by microscopy , as well as enterocytozoon bieneusi , are zoonotic ( mansfield and gajadhar , 2004 , ye et al . , 2012 , giardia duodenalis is a particularly zoonotic parasitic protozoan that infects a wide range of mammals , including nhps ( feng and xiao , 2011 ) . animals are infected when they ingest food or water contaminated with giardia cysts ( graczyk et al . , 2003 ) . the assemblage b were the nhps host - adaptated , in 96.7% ( 118/122 ) of the positive isolates , which were zoonotic assemblage ( karim et al . , 2015a ) . are usually associated with intestinal pathology , resulting in diarrhea in both humans and animals ( ryan and hijjawi , 2015 ) . they are transmitted via the fecal - oral route by either direct contact or the ingestion of contaminated food or water . the protozoan can disperse rapidly because they have a monoxenous life cycle , a low infective dose , and a short prepatent period ( graczyk et al . however , the prevalence rate found in this study is much lower than that found in sri lanka ( 27.2% , 27/125 ) ( ekanayake et al . , 2006 ) and e. bieneusi is a common parasitic pathogen in nhps with high prevalence ( 16.3% ) which difficult to detect by light microscopy . a lower infection rate ( 12.3% ) of e. bieneusi was reported in kenya ( li et al . , 2011 ) , while higher infection rates ( 28.2% and 18.5% ) were found in free - range macaque monkeys in guizhou and cynomolgus monkeys in guangxi , china , respectively ( ye et al . , 2012 , ye et al . , 2014 ) . altogether , 34 e. bieneusi its genotypes were found involving 26 genotypes ( 263/306 , 86.0% ) belonged to group 1 with zoonotic potential ( karim et al . thus , the genotypes in nhps had zoonotic potential , and nhps could act as reservoirs of human microsporidiosis . the entamoeba spp . had the highest infection rate ( 36.4% ) by microscopy , and was observed in the majority of the nhp species ( 25/34 ) examined ( table 1s ) . they are also known to be a highly prevalent intestinal parasite in ethiopia , uganda , senegal , tanzania , italian , cameroon , etc ( legesse and erko , 2004 , gillespie et al . , 2005 , petrov et al . , 2010 , are human pathogens that are transmitted by various forms of contact due to their direct life cycle ( pedersen et al . , 2005 , although , only e. dispar and e. coli were found in this study ( unpublished data ) which were non - pathogenic species with low risk of zoonotic transmission from nhps to human , the zoonotic transmit also should be pay attention . are obligate intracellular parasites that inhabit the bile duct or intestinal mucosal epithelial cells of various vertebrates ( legua and seas , 2013 ) . until now , 1999 , ortega and sanchez , 2010 , li et al . , 2015b ) and one in humans ( zhou et al . , 2011 ) . the highest prevalence of cyclospora spp . were found in ethiopia ( 22.0% , 13/59 ) ( legesse and erko , 2004 ) . and , cyclospora - like organisms were also detected in monkeys ( zhao et al . the helminths , including trichuris spp . , strongyloides spp . , ascaris spp . , , are parasitic with a high potential for transmission to humans because of their simple life cycles . they have been reported in several populations of primates ( ocaido et al . , 2003 , legesse and erko , 2004 , gillespie et al . , 2005 , bezjian et al . , 2008 , ( table 1s ) , in contrast to the colobus monkeys in cte divoire ( kouassi et al . , 2015 ) . unfortunately , it is difficult to identify the helminths ' species only based on morphology of oocysts or eggs . a comprehensive study of their genetic diversity is necessary to confidently distinguish the species and genotypes of these intestinal parasites . in conclusion , this is an investigation of the parasites in nhps in china , which detailed parasites infection status and reviewed of molecular characterization of four intestinal protozoans . this baseline parasitological data provides important information for the elimination of such parasites and monitor the potential transmission of zoonotic infections from nhps .

backgroundnanoemulsions consist of very stable nanodroplets of oil dispersed in an aqueous phase , typically below 300 nm in size . they can be used to obtain a very fine , homogeneous dispersion of lipophilic compounds in water , thus facilitating their handling and use in nanomedicine . however , the drawback is that they are suspended in an aqueous media . this study proposes a novel technique for drying lipid nanoemulsion suspensions to create so - called trojan particles , ie , polymer microparticles ( around 2 m ) which very homogeneously entrap the nano - oil droplets ( around 150 nm ) in their core.methodsmicroencapsulation of the nanoemulsions was performed using a spray - drying process and resulted in a dried powder of microparticles . by using a low - energy nanoemulsification method and relatively gentle spray - drying , the process was well suited to sensitive molecules . the model lipophilic molecule tested was vitamin e acetate , encapsulated at around 20% in dried powder.resultswe showed that the presence of nanoemulsions in solution before spray - drying had a significant impact on microparticle size , distribution , and morphology . however , the process itself did not destroy the oil nanodroplets , which could easily be redispersed when the powder was put back in contact with water . high - performance liquid chromatography follow - up of the integrity of the vitamin e acetate showed that the molecules were intact throughout the process , as well as when conserved in their dried form.conclusionthis study proposes a novel technique using a spray - drying process to microencapsulate nanoemulsions . the multiscale object formed , so - called trojan microparticles , were shown to successfully encapsulate , protect , and release the lipid nanodroplets .

microencapsulation processes are generally used for both isolating and protecting sensitive substances or bioactive molecules and facilitating their controlled release.1 the formulation of microparticles is generally tailored to suit the target administration route . microparticles can be built with a core / shell structure composed of two materials , ie , so - called core materials and wall materials . active ingredients are either entrapped in the core materials , or form the core material itself , enclosed and protected by the wall material . the different formulation processes used to produce microparticles are divided into three groups , 1 ) chemical methods , eg , polymerization or interface polycondensation ; 2 ) physicochemical methods , such as coacervation , solvent diffusion , solvent evaporation , phase separation , or rapid expansion of supercritical fluids ; and 3 ) physicomechanical methods , including spray - drying , fluidized - bed technology , or pan coating.2 in this paper , we focus on the third group of methods and notably on use of the spray - drying process in the formulation of microparticles . spray - drying is a widely used technology for microencapsulation in the food and pharmaceutical industries . the process rapidly converts a liquid ( which can be a solution , a suspension , or an emulsion ) into a dried powder . core materials , homogeneously solubilized or dispersed in an aqueous media , are sprayed in contact with a hot gas flux . the sprayed droplets are still quite small , ranging from a few to a few hundred micrometers , thus developing a large air/ water interface area , resulting in effective heat transfer . as a result , the drying process is complete when the temperature of the particle is equal to that of the air.3 at this point , the active ingredients appear homogenously dispersed in the matricial , dried , microparticulate structure.4 spraying and drying the particles is a rapid process , performed within a few seconds . it is this speed that makes the process suitable for use with sensitive or thermosensitive compounds without resulting in their degradation . when this process is performed with biodegradable and biocompatible wall materials , and with adapting the particle sizes and densities to the administration route , spray - drying has been shown to be suitable for the inhalation route5,6 and intraocular administration.7 the solid forms generated can also be used to make tablets or capsules for oral administration , enhancing intestinal absorption and bioavailability . the nature of the solid formed helps increase the stability of compounds liable to oxidize in solution . such micrometric systems present many advantages for pharmaceutical applications , such as easy manipulation and administration of the microparticles , for instance , and reduced clearance after subcutaneous or intraocular administration . however , a major disadvantage is their low penetration and diffusion in the tissues . to counter this problem , these multiscaled systems are known as trojan particles.7 when dried or in suspension , nanoparticles are not usable due to their low stability , aggregation , and hydrolysis in water . their manipulation is greatly improved when encapsulated in the aforementioned trojan systems . however , to date , only recent work proposes such formulations , using only polymeric nanoparticles.79 although used as drug carriers , these polymeric nanoparticles present ongoing drawbacks due to the polymers themselves , which limit the drug encapsulation rates or leave polymeric residues in living tissues . the innovative concept of our work here is the creation of trojan microparticles by spray - drying and encapsulation , not of polymeric nanoparticles but of nanoemulsions , which are fundamentally different systems . for pharmaceutical applications , nanoemulsions prove to be extremely stable systems not only in suspension before spray - drying , but also when the droplets are conserved through the process in water after dissolution of the powder . the core of the droplet is often only made up of active ingredients ( as is the case here ) , and thus the encapsulation rates and yields need to be a few orders of magnitude higher than for polymeric nanoparticles . for instance , gomez - gaete et al7 encapsulated bioactive hydrophobic molecules of dexamethasone at nanoparticle encapsulation rates as low as 0.7% , and when the optimized solubilization of lipophilic compounds in oils increased , this rate and the direct emulsification of liquid lipids gave a full encapsulation of 100% . such innovative trojan particles also provide solutions for drying lipids or lipophilic bioactive molecules , and their nanoscaled formulation increases and optimizes their bioavailability at the administration site . the major potential problem concerning microencapsulation of nanoemulsions , which we propose to address here , is conservation of the nanoemulsion structure and size distribution throughout the spray - drying process . nanoemulsions are generally defined as oil - in - water emulsion droplets , with a size ranging in the nanometric scale , typically 20300 nm.1012 owing to their very small size , nanoemulsions have great stability for up to several months . the reasons for this stability are well documented in the literature,1315 and originate in the unlikeliness of the droplets to flocculate and coalesce . , nanoemulsions can be used as a new tool for chemical and pharmaceutical applications , for instance providing a homogeneous dispersion of otherwise poorly soluble materials in water . nanoemulsions can be used as nanocarriers for drugs or contrast agents in the applications of targeting and controlled delivery . as regards the formulation processes for nanoemulsions , they can be divided into two groups , ie , high - energy and low - energy methods.13,14 high - energy methods use specific devices to form nanoemulsions , such as high - pressure homogenizers or ultrasound generators , whereas low - energy methods make the most of the intrinsic physicochemical properties of the components in the formulation to attain the nanometric range . in this case , nanoemulsions are formed spontaneously at room temperature.14 in our work , we have chosen to use low - energy methods , which are also both solvent - free and polymer - free , for the generation of a definitively stable oil suspension dispersed in an aqueous phase.1315 it is worth noting the many studies done on microencapsulating of emulsions using spray - drying.1622 however , the objectives and scope of these published works are restricted to the drying of oily or fatty species , and do not deal with the conservation of the structure ( eg , size ) of the dispersed system or emulsified forms . recent works have reported very efficient methods obtaining up to 90% of oil content in the dried powder.23 however , the emulsion droplet size plays an important role in the encapsulation efficiency during spray drying,1618 ie , the smaller the size of the encapsulated emulsion , the more efficient the encapsulation . in this respect , the microencapsulation of nanoemulsions can further enhance the result without using specific interface crosslinking technology.23 returning to the main objective , ie , the formulation of trojan particles , our aim is to conserve the nanometric scale of the encapsulated droplets entrapped in the microparticles and to ensure their delivery , with resolubilization of the dried microparticle powder in water . in this work this should open new doors for the application of these simple and promising nanoparticulate systems . low - energy nanoemulsification methods performed at room temperature , associated with the gentle process of spray - drying , make the whole formulation process suitable for encapsulation of sensitive molecules . in this study , we used a formulation with vitamin e acetate as the model lipophilic molecule to be encapsulated . this molecule constitutes the hydrophobic phase of the formulation and , consequently , the nanoemulsion core was made up exclusively of vitamin e acetate . the sample characterization was obtained by scanning electron microscopy ( sem ) , and the particle size distributions were worked out by analyzing the sem pictures , thus allowing a quantitative comparison of the powders . the microencapsulation process was first evaluated as a function of the various formulation parameters , such as the nature of the wall materials and the concentration in solution before spray - drying . these preliminary results subsequently allowed us to optimize the microencapsulation of a vitamin e acetate nanoemulsion . a number of biodegradable polymers were evaluated for use as wall materials in the process , namely , gum arabic , whey protein , polyvinyl alcohol , modified starches , maltodextrin , hydroxypropyl beta cyclodextrins , and pregelatinized hydroxypropyl pea starch . the stability of the nanoemulsions was controlled throughout the process by dynamic light scattering , and the integrity and stability of vitamin e acetate molecules was followed for 3 months using high - performance liquid chromatography ( hplc ) . vitamin e acetate ( dl - alpha - tocopheryl acetate ) was purchased from sigma ( st louis , mo ) . nonionic surfactants from basf ( ludwigshafen , germany ) , ie , cremophor elp ( polyoxiethylated-35 castor oil , hydrophilic lipophilic balance approximately 1214 ) were kindly provided by laserson ( etampes , france ) , and used as received . the wall materials selected were gum arabic ( cni colloides naturels international , rouen , france ) , whey protein ( davisco foods international inc , eden prairie , mn ) , cleargum co 03 ( modified starch , roquette , lestrem , france ) , glucidex it 12 ( maltodextrin , de 12 , roquette ) , polyvinyl alcohol ( pva , sigma ) , kleptose hpb parenteral grade ( hydroxypropyl beta cyclodextrins , roquette ) , nutriose fb 06 ( modified starch , roquette ) , and lycoat rs 780 ( pregelatinized hydroxypropyl pea starch , roquette ) . finally , ultrapure water was obtained using the milliq filtration system ( millipore , saint - quentin - en - yvelines , france ) . the samples were composed of nanoemulsions dispersed in an aqueous solution to dissolve the wall materials . vitamin e acetate nanoemulsions were selected as model suspensions with a controllable size and polydispersity . nanoemulsions were formulated according to the low - energy emulsification process described elsewhere.14 in short , nanoemulsion droplets were spontaneously formed after bringing the two phases into contact . the first was composed of vitamin e acetate ( the oily phase ) plus the hydrophilic surfactant , both totally miscible in each other and gently homogenized at room temperature , and the second phase was pure water . once these two liquid phases were mixed , the hydrophilic species were immediately solubilized by the aqueous phase , inducing the demixing of the oil ( through spinodal decomposition ) in the form of nanometric emulsion droplets . vitamin e acetate was chosen as a model oily phase , mixed with the nonionic surfactants , and then brought into contact with the aqueous phase , thus generating nanoemulsions . the spontaneous emulsification processes were characterized , as described previously,14 by following the impact of the surfactant oil weight ( sow ) ratio ( wsurfactant/[wsurfactant + woil ] 100 ) on both the hydrodynamic diameter and polydispersity index . the optimized formulation is a compromise between nanoemulsions presenting a good monodispersity , a size in the nanometric range , and the lowest possible amount of surfactant . the water added for the generation of nanoemulsions influences neither their physicochemical properties nor their size and polydispersity index . the size distribution and polydispersity of the nanoemulsions were assessed by dynamic light scattering using a malvern nano zs instrument ( malvern , orsay , france ) . a heliumneon laser ( 4 mw ) was operated at 633 nm with the scatter angle fixed at 173 , and the temperature was maintained at 25c . the polydispersity index is a measure of the broadness of the size distribution derived from the cumulative analysis of dynamic light scattering . for a single gaussian population with standard deviation , and mean size xpcs , the polydispersity index indicates the quality of the dispersion , from values lower than 0.1 for acceptable measurements and good quality colloidal suspensions , to values close to 1 for poor quality samples , either with droplet sizes out of the colloidal range or with a very high polydispersity . measurements were performed in triplicate , before and after the spray - drying process ( filtered at 0.45 m in the above case ) . as regards the polymers used as wall materials , these were dissolved and swelled in ultrapure water 1 day prior to performing the experiments . the amount of wall material and its impact on the spray - dried particles was studied , as were the respective proportions of core and wall materials which also appear to be an important parameter of the formulation . these parameters were studied and optimized in order to incorporate the highest possible amount of oil with the dried , powdery aspect of the samples . the wall material solution was incorporated in the nanoemulsions of vitamin e acetate under stirring until a homogenous suspension was obtained . the liquid suspensions were atomized into powders by the bchi mini spray dryer b-290 ( flawil , switzerland ) . the two - fluid nozzle dispersed the liquid suspensions into thin droplets , which were heated in the drying chamber . the speed of the process is acknowledged as being well suited to the spray - drying of sensitive or thermosensitive molecules . the operational conditions were as follows : aspiration rate 100% ; inlet temperature 150c ; and solution feed rate 10 ml / minute . the size distribution and morphology of the spray - dried microparticles were evaluated by sem ( philips xl20 , university of strasbourg ; philips , amsterdam , the netherlands ) . the specimen was fixed on a carbon support , coated with a palladium layer , and analyzed at 20 kv . the particle size distributions were analyzed from sem micrographs using a software - assisted method previously developed in our laboratory and already described elsewhere.24 the exact number of particles and their diameters were calculated from raw images , even for agglomerated particles . hence , even if particles form aggregates , our method isolates each particle from the rest and defines its surface area , thus allowing individual identification and disclosing the particle size distribution . a log - normal extrapolation was applied , with a probability density function of the form f ( x ; , ) = exp ( [ln x ]/2)/(x[2 ] ) ( where and are the mean and standard deviation , respectively ) , thus making it possible to do a quantitative comparison of the sem pictures and consequently enabling us to compare the spray - dried samples . the powders are formulated after carefully selecting a wall material / nanoemulsion ratio where no aggregates are formed so that the sample remains in powdered form even after drying . for conventional imaging in sem , the sample needs to be fixed on a carbon support followed by coating with a conductive metallic layer , which makes the particles appear as if they are aggregated . however , the advantage of the software used for analyzing the size distribution lies in its ability to distinguish single particles even when they look aggregated . the originality of this approach lies in the possibility of quantitative analysis of the sem pictures and thus of sample comparison . each specimen of wall material encapsulating the nanoemulsions was spray - dried twice and the sem measurements for each one were performed on three distinct sem carbon supports . for each given experiment , a representative picture of the sem results obtained was selected and reported in figures accompanying this paper . hplc was used ( pump lc-126 , detector lc-168 , and injector lc-508 ; beckman - coulter , fullerton , ca ) to follow the stability and integrity of vitamin e acetate encapsulated in the spray - dried microparticles . the operational conditions were defined as : stationary phase , column c8 , length 0.125 m , 4.0 mm ( cc125/4 lichrospher 100 - 5 rp-8 ; macherey - nagel , dren , germany ) ; mobile phase , a mixture of 50 vol of acid aqueous phase ( ph 2.3 ) and 950 vol of a mixture of 85% methyl alcohol and 15% isopropanol ; flow rate 1.2 ml / minute ; and a spectrophotometer operated at 285 nm and injection volume 20 l . the reference samples of vitamin e acetate were selected at a concentration of 89 g / ml . quantification of the vitamin e acetate trapped in the spray - dried microparticles was performed by collecting an amount of powder with a vitamin acetate content identical to that of the reference sample . the powder was then dissolved in the mixture of water and isopropanol . however , in accordance with the wall material properties , different ratios between water and isopropanol were used , adapted to the wall materials . the amount of vitamin e acetate was determined at initial time ( ie , immediately after the spray - drying process ) and then once a week for each wall material over 3 months . each specimen of wall material was spray - dried twice and the hplc measurements for each powder were carried out in triplicate . vitamin e acetate ( dl - alpha - tocopheryl acetate ) was purchased from sigma ( st louis , mo ) . nonionic surfactants from basf ( ludwigshafen , germany ) , ie , cremophor elp ( polyoxiethylated-35 castor oil , hydrophilic lipophilic balance approximately 1214 ) were kindly provided by laserson ( etampes , france ) , and used as received . the wall materials selected were gum arabic ( cni colloides naturels international , rouen , france ) , whey protein ( davisco foods international inc , eden prairie , mn ) , cleargum co 03 ( modified starch , roquette , lestrem , france ) , glucidex it 12 ( maltodextrin , de 12 , roquette ) , polyvinyl alcohol ( pva , sigma ) , kleptose hpb parenteral grade ( hydroxypropyl beta cyclodextrins , roquette ) , nutriose fb 06 ( modified starch , roquette ) , and lycoat rs 780 ( pregelatinized hydroxypropyl pea starch , roquette ) . finally , ultrapure water was obtained using the milliq filtration system ( millipore , saint - quentin - en - yvelines , france ) . the samples were composed of nanoemulsions dispersed in an aqueous solution to dissolve the wall materials . vitamin e acetate nanoemulsions were selected as model suspensions with a controllable size and polydispersity . nanoemulsions were formulated according to the low - energy emulsification process described elsewhere.14 in short , nanoemulsion droplets were spontaneously formed after bringing the two phases into contact . the first was composed of vitamin e acetate ( the oily phase ) plus the hydrophilic surfactant , both totally miscible in each other and gently homogenized at room temperature , and the second phase was pure water . once these two liquid phases were mixed , the hydrophilic species were immediately solubilized by the aqueous phase , inducing the demixing of the oil ( through spinodal decomposition ) in the form of nanometric emulsion droplets . vitamin e acetate was chosen as a model oily phase , mixed with the nonionic surfactants , and then brought into contact with the aqueous phase , thus generating nanoemulsions . the spontaneous emulsification processes were characterized , as described previously,14 by following the impact of the surfactant oil weight ( sow ) ratio ( wsurfactant/[wsurfactant + woil ] 100 ) on both the hydrodynamic diameter and polydispersity index . the optimized formulation is a compromise between nanoemulsions presenting a good monodispersity , a size in the nanometric range , and the lowest possible amount of surfactant . the water added for the generation of nanoemulsions influences neither their physicochemical properties nor their size and polydispersity index . the size distribution and polydispersity of the nanoemulsions were assessed by dynamic light scattering using a malvern nano zs instrument ( malvern , orsay , france ) . a heliumneon laser ( 4 mw ) was operated at 633 nm with the scatter angle fixed at 173 , and the temperature was maintained at 25c . the polydispersity index is a measure of the broadness of the size distribution derived from the cumulative analysis of dynamic light scattering . for a single gaussian population with standard deviation , and mean size xpcs , the polydispersity index indicates the quality of the dispersion , from values lower than 0.1 for acceptable measurements and good quality colloidal suspensions , to values close to 1 for poor quality samples , either with droplet sizes out of the colloidal range or with a very high polydispersity . measurements were performed in triplicate , before and after the spray - drying process ( filtered at 0.45 m in the above case ) . as regards the polymers used as wall materials , these were dissolved and swelled in ultrapure water 1 day prior to performing the experiments . the amount of wall material and its impact on the spray - dried particles was studied , as were the respective proportions of core and wall materials which also appear to be an important parameter of the formulation . these parameters were studied and optimized in order to incorporate the highest possible amount of oil with the dried , powdery aspect of the samples . the wall material solution was incorporated in the nanoemulsions of vitamin e acetate under stirring until a homogenous suspension was obtained . the liquid suspensions were atomized into powders by the bchi mini spray dryer b-290 ( flawil , switzerland ) . the two - fluid nozzle dispersed the liquid suspensions into thin droplets , which were heated in the drying chamber . the speed of the process is acknowledged as being well suited to the spray - drying of sensitive or thermosensitive molecules . the operational conditions were as follows : aspiration rate 100% ; inlet temperature 150c ; and solution feed rate 10 ml / minute . the size distribution and morphology of the spray - dried microparticles were evaluated by sem ( philips xl20 , university of strasbourg ; philips , amsterdam , the netherlands ) . the specimen was fixed on a carbon support , coated with a palladium layer , and analyzed at 20 kv . the particle size distributions were analyzed from sem micrographs using a software - assisted method previously developed in our laboratory and already described elsewhere.24 the exact number of particles and their diameters were calculated from raw images , even for agglomerated particles . hence , even if particles form aggregates , our method isolates each particle from the rest and defines its surface area , thus allowing individual identification and disclosing the particle size distribution . a log - normal extrapolation was applied , with a probability density function of the form f ( x ; , ) = exp ( [ln x ]/2)/(x[2 ] ) ( where and are the mean and standard deviation , respectively ) , thus making it possible to do a quantitative comparison of the sem pictures and consequently enabling us to compare the spray - dried samples . the powders are formulated after carefully selecting a wall material / nanoemulsion ratio where no aggregates are formed so that the sample remains in powdered form even after drying . for conventional imaging in sem , the sample needs to be fixed on a carbon support followed by coating with a conductive metallic layer , which makes the particles appear as if they are aggregated . however , the advantage of the software used for analyzing the size distribution lies in its ability to distinguish single particles even when they look aggregated . the originality of this approach lies in the possibility of quantitative analysis of the sem pictures and thus of sample comparison . each specimen of wall material encapsulating the nanoemulsions was spray - dried twice and the sem measurements for each one were performed on three distinct sem carbon supports . for each given experiment , a representative picture of the sem results obtained was selected and reported in figures accompanying this paper . hplc was used ( pump lc-126 , detector lc-168 , and injector lc-508 ; beckman - coulter , fullerton , ca ) to follow the stability and integrity of vitamin e acetate encapsulated in the spray - dried microparticles . the operational conditions were defined as : stationary phase , column c8 , length 0.125 m , 4.0 mm ( cc125/4 lichrospher 100 - 5 rp-8 ; macherey - nagel , dren , germany ) ; mobile phase , a mixture of 50 vol of acid aqueous phase ( ph 2.3 ) and 950 vol of a mixture of 85% methyl alcohol and 15% isopropanol ; flow rate 1.2 ml / minute ; and a spectrophotometer operated at 285 nm and injection volume 20 l . the reference samples of vitamin e acetate were selected at a concentration of 89 g / ml . quantification of the vitamin e acetate trapped in the spray - dried microparticles was performed by collecting an amount of powder with a vitamin acetate content identical to that of the reference sample . the powder was then dissolved in the mixture of water and isopropanol . however , in accordance with the wall material properties , different ratios between water and isopropanol were used , adapted to the wall materials . the amount of vitamin e acetate was determined at initial time ( ie , immediately after the spray - drying process ) and then once a week for each wall material over 3 months . each specimen of wall material was spray - dried twice and the hplc measurements for each powder were carried out in triplicate . first , all the different wall materials were individually spray - dried in order to evaluate their potential influence on the spray - drying process and on the microparticle properties , ie , size , polydispersity , and morphology . the samples were spray - dried at 15 wt% in water before processing ( a representative concentration ) . the sem pictures are presented in figure 1 , including the quantitative study of the size maxima and standard deviation values . the results are not only significant for size distribution and polydispersity , but also for particle shape and morphology , which are closely linked to the interface properties of the compounds and to the spray - drying process . the first observation shows that the microparticle sizes are quite small , generally 14 m . furthermore , fluctuations of the maxima between different species are relatively reduced and not significantly different . however , differences do exist , and could be due to the surfactant properties of the polymers , ie , gum arabic , whey protein , maltodextrin , and polyvinyl alcohol , giving rise to smaller particles , whereas kleptose , nutriose , cleargum , and lycoat produced bigger ones . although the samples appear globally homogeneous regardless of the materials used and the microparticles show smooth surfaces and spherical geometry , some of them ( eg , figures 1b , 1c , 1d , 1f , and 1 g ) show intense invaginations , with a shape resembling a deflated ball . to explain this result , an interesting parallel can be drawn between these deflated shapes and the global microparticulate sizes , in that the deflated shapes tend to correspond to the bigger particles . this is understandable because before drying all the wall material concentrations in water were the same and the droplets began to dry at their periphery . this is clearly illustrated by the hydroxypropyl beta cyclodextrin particles ( kleptose ) in figure 1c , in which the bigger specimens have shapes with very thin shells . these particular morphologies appear linked to the size of the sprayed droplets , ( ie , after spraying and before drying ) and thus to the surface properties of the wall material solutions . conversely , polyvinyl alcohol , known for its surfactant properties , forms spherical microparticles ( figure 1h ) . the picture also shows the presence of polymer wires that physically link the particles , their formation being influenced by interface phenomena and low surface tensions . this series of experiments was designed to observe the effect of the polymer concentration in the solution on the properties and morphology of the spray - dried microparticles . we thus studied the representative case of gum arabic solutions at 5 , 10 , 15 , 20 , and 30 wt% . the results are presented in figure 3 , including the quantitative study of the size maxima and standard deviation values . sizes and standard deviations appear very similar in all the concentrations , ranging from 1.5 and 2.0 m , with standard deviations from 0.8 and 1.6 m . however , there is a significant difference in the samples as regards global aspect , particle shape , and homogeneity . the higher the polymer concentration , the larger the size distribution , reaching huge particle sizes larger than 10 m ( shown in figures 3d and 3e ) , which are in fact not visible in the statistical analysis because their proportions within the whole population are very small . this is probably due to the higher viscosity of the suspension , which increases with the concentration . accordingly , the representative wall material concentration of 15 wt% was chosen for the rest of the experiments . as described in the methods section , the nanoemulsification process is characterized by studying the influence of the surfactant - to- oil ratio on the size and polydispersity of the nanoemulsions . the general trend , as expected and found with various other systems,14 shows a lowering of the droplet size and polydispersity index as the amount of surfactant is increased ( ie , an increase of the surfactant - to - oil ratio ) . the first results highlight the efficiency of the nanoemulsification process : they are all the more significant because the formulation proposed here uses only vitamin e acetate as a hydrophobe , totally substituting any oily compounds generally used in these spontaneous emulsification processes ( eg , oleic macrogols or triglycerides ) . nanoemulsions with relatively monodispersed droplets ( polydispersity index < 0.2 ) , with dh = 189 nm , are obtained from a surfactant to oil ratio of 20% . with a surfactant to oil ratio of 40% , the droplet size falls to below 100 nm . in order to ensure efficient nanoemulsion encapsulation of the spray - dried microparticles , the present proof of concept study used nanodroplets below 100 nm . hence , the chosen value was a surfactant to oil of 40% ( indicated with the arrow in figure 4 ) , giving rise to nanoemulsions exhibiting a hydrodynamic diameter dh of 84 nm and a polydispersity index of 0.11 . the production of dried microparticles to entrap the oily nanoemulsion droplets was performed , as described earlier , by spray - drying the nanoemulsion droplets in suspension in an aqueous bulk phase , thereby solubilizing the wall material polymers . the impact of the oil + surfactant / wall material weight ratio on the microparticulate properties , morphology , and size distribution was evaluated for the representative case of whey protein . the results are reported in figure 5 , and the quantitative study of the size maxima and standard deviation values are reported in figure 6 . four different ratios were tested , ie , 1:1 , 1:2 , 1:3 , and 1:4 , corresponding to vitamin e acetate loadings of 37.5 , 27.3 , 21.4 , and 17.6 wt% , respectively . the samples appear to be very homogenous , generally presenting a similar aspect . when compared with the pure formulations shown in figure 1a , the first point to observe is the almost perfect spherical shape of the particles . the deflated morphology of the particles discussed above is no longer present , now being replaced by spherical shapes and smooth surfaces . this could be due to the global increase of droplet loading before drying , which is doubled with the ratio 1:1 . hence , the empty cavities formed with pure wall materials no longer exist , giving way to spherical morphologies . as for the size distribution , a clear difference is observed between the ratios 1:1 , 1:2 , 1:3 , and 1:4 . looking at this result in terms of interface properties , we can note that not only do the nanoemulsions here show significant surfactant properties , but that the droplet loading is reduced with the ratio of 1:4 , thus affording a decrease in the particle size . furthermore , these results were obtained with whey protein , for which each ratio gave rise to powdery and homogeneous samples . however , using the other wall materials , this limit fluctuated from 1:1 to 1:4 . wall materials were spray - dried along with surfactant alone , vitamin e acetate alone , and both the surfactant and the vitamin e acetate formulated as a nanoemulsion . for each experiment using surfactant alone and vitamin e acetate alone , the compound amounts strictly corresponded to those required for nanoemulsion formulation . the aim of these experiments was to see whether the nanodispersed forms themselves could have an influence on the spray - drying process . the results are presented in figure 7 for representative wall materials , including quantitative study of the size maxima and standard deviation values for gum arabic ( figure 7b ) , whey protein ( figure 7c ) , and kleptose and nutriose ( figure 7d ) , and are summarized in figure 8 . here , the first observation concerns the morphology of the microparticles as a function of the compounds added to the wall materials . as discussed earlier , the deflated aspect of the particles is generally inhibited by the presence of the oily phase or nanoemulsions , but , conversely , it seems to be enhanced by the surfactant ( except with the whey protein for which this deflated effect appears less pronounced ) . indeed , for a similar amount of surfactants using the wall material + surfactant alone system and the wall material + nanoemulsion system , the resulting microparticle sizes were significantly different , especially for gum arabic and whey protein . however , even if this result is not as apparent with kleptose and nutriose , the global trend highlighted in figure 8 shows that generally the size is slightly reduced with the wall material + surfactant alone system and slightly increased for the wall material + vitamin e acetate alone system . these results corroborate the idea that nanoformulated materials can present more enhanced effects than those induced by nonformulated compounds . in our case , only the surfactant is likely to affect the interface phenomena governing the spray - drying process . this can be illustrated by comparison of the wall material + surfactant alone system and the wall material + nanoemulsion system . this suggests that some of the nanoemulsion droplets are trapped at the water / air interface , reducing the surface tension even more than with the surfactant alone , and giving rise to smaller droplets . one objective of this study was to prove that it was possible to microencapsulate nanoemulsion droplets in order to propose new solutions for the delivery of nanoemulsions , thus allowing enhanced absorption of the encapsulated compounds . the microencapsulation of nanoemulsion droplets also requires that , once entrapped in the dried microparticles , the nanoemulsion oil droplets conserve their size and individual nature . the polymer is rapidly solubilized due to the huge specific surface of the powder and , if kept intact during the process , the nanoemulsion droplets can be redispersed in water . their resolubilization in water allowed redispersion of the nanoemulsion droplets . because all the wall materials used are soluble in water , the times within which the nanoemulsions are redispersed in water appear to be relatively short , ie , a few minutes . they will depend on both the water / particle ratio and the nature of the wall materials ( ie , on the kinetics of the wall material solubilization in water ) . for instance , for a given amount of powders mixed in water ( 10 wt% ) , redispersion of the nanoemulsions was achieved for the different wm as follows : gum arabic about 5 minutes ; whey protein about 5 minutes ; kleptose about 3 minutes ; and nutriose about 3 minutes . after redispersion , the samples , like typical nanometric dispersions , were translucent and their characterization was performed by dynamic light scattering . the results are reported in table 1 , presenting the hydrodynamic diameter and polydispersity index of the nanoemulsion droplets , just after formulation ( called raw nanoemulsions ) , after mixing with wall materials before and after spray - drying , and redispersion in the same volume of water . to summarize , a comparison of the samples before and after spray - drying showed a significant increase in size , and was generally doubled for all the samples . this results from slight droplet coalescence and can simply be due to turbulence and heat during the spray - drying process . the results also vary in the function of the wall materials , again indicating that the oil / polymer interactions and affinities could play a role in droplet merging . nevertheless , the average size of the redispersed droplets remained at about 150 nm and , with a polydispersity index of around 0.2 , such a suspension can still rightly be considered a nanoemulsion . taking into account all the results of the present study , a schematic illustration of the microparticulate structure is shown in figure 9 . these multiscaled systems can in fact be used as carriers for nanoemulsions , ensuring their easy manipulation , concentration , and administration . stability studies were performed on the powders , the results of which are reported in figure 10 . we can first note that , at initial time , the measurements show approximately 100% of vitamin e acetate for all the seven wall materials tested . this simply indicates that the spray - drying process does not destroy the encapsulated molecules . it is also worth noting that only one peak of vitamin e acetate was observed for all the wall materials studied and that no other peaks appeared over 3 months . in other words this result is confirmed and corroborated by the overall aspect of the curves in figure 10 , indicating a constant amount of vitamin e acetate with a recovery of approximately 100% . these results confirm that the spray - drying process can be considered an efficient method for the microencapsulation of nanoemulsions , ensuring the integrity of the molecules throughout the process , as well as their conservation during storage of the dried product . first , all the different wall materials were individually spray - dried in order to evaluate their potential influence on the spray - drying process and on the microparticle properties , ie , size , polydispersity , and morphology . the samples were spray - dried at 15 wt% in water before processing ( a representative concentration ) . the sem pictures are presented in figure 1 , including the quantitative study of the size maxima and standard deviation values . the results are not only significant for size distribution and polydispersity , but also for particle shape and morphology , which are closely linked to the interface properties of the compounds and to the spray - drying process . the first observation shows that the microparticle sizes are quite small , generally 14 m . furthermore , fluctuations of the maxima between different species are relatively reduced and not significantly different . however , differences do exist , and could be due to the surfactant properties of the polymers , ie , gum arabic , whey protein , maltodextrin , and polyvinyl alcohol , giving rise to smaller particles , whereas kleptose , nutriose , cleargum , and lycoat produced bigger ones . although the samples appear globally homogeneous regardless of the materials used and the microparticles show smooth surfaces and spherical geometry , some of them ( eg , figures 1b , 1c , 1d , 1f , and 1 g ) show intense invaginations , with a shape resembling a deflated ball . to explain this result , an interesting parallel can be drawn between these deflated shapes and the global microparticulate sizes , in that the deflated shapes tend to correspond to the bigger particles . this is understandable because before drying all the wall material concentrations in water were the same and the droplets began to dry at their periphery . this is clearly illustrated by the hydroxypropyl beta cyclodextrin particles ( kleptose ) in figure 1c , in which the bigger specimens have shapes with very thin shells . these particular morphologies appear linked to the size of the sprayed droplets , ( ie , after spraying and before drying ) and thus to the surface properties of the wall material solutions . conversely , polyvinyl alcohol , known for its surfactant properties , forms spherical microparticles ( figure 1h ) . the picture also shows the presence of polymer wires that physically link the particles , their formation being influenced by interface phenomena and low surface tensions . this series of experiments was designed to observe the effect of the polymer concentration in the solution on the properties and morphology of the spray - dried microparticles . we thus studied the representative case of gum arabic solutions at 5 , 10 , 15 , 20 , and 30 wt% . the results are presented in figure 3 , including the quantitative study of the size maxima and standard deviation values . sizes and standard deviations appear very similar in all the concentrations , ranging from 1.5 and 2.0 m , with standard deviations from 0.8 and 1.6 m . however , there is a significant difference in the samples as regards global aspect , particle shape , and homogeneity . the higher the polymer concentration , the larger the size distribution , reaching huge particle sizes larger than 10 m ( shown in figures 3d and 3e ) , which are in fact not visible in the statistical analysis because their proportions within the whole population are very small . this is probably due to the higher viscosity of the suspension , which increases with the concentration . accordingly , the representative wall material concentration of 15 wt% was chosen for the rest of the experiments . as described in the methods section , the nanoemulsification process is characterized by studying the influence of the surfactant - to- oil ratio on the size and polydispersity of the nanoemulsions . the general trend , as expected and found with various other systems,14 shows a lowering of the droplet size and polydispersity index as the amount of surfactant is increased ( ie , an increase of the surfactant - to - oil ratio ) . the first results highlight the efficiency of the nanoemulsification process : they are all the more significant because the formulation proposed here uses only vitamin e acetate as a hydrophobe , totally substituting any oily compounds generally used in these spontaneous emulsification processes ( eg , oleic macrogols or triglycerides ) . nanoemulsions with relatively monodispersed droplets ( polydispersity index < 0.2 ) , with dh = 189 nm , are obtained from a surfactant to oil ratio of 20% . with a surfactant to oil ratio of 40% , the droplet size falls to below 100 nm . in order to ensure efficient nanoemulsion encapsulation of the spray - dried microparticles , the present proof of concept study used nanodroplets below 100 nm . hence , the chosen value was a surfactant to oil of 40% ( indicated with the arrow in figure 4 ) , giving rise to nanoemulsions exhibiting a hydrodynamic diameter dh of 84 nm and a polydispersity index of 0.11 . the production of dried microparticles to entrap the oily nanoemulsion droplets was performed , as described earlier , by spray - drying the nanoemulsion droplets in suspension in an aqueous bulk phase , thereby solubilizing the wall material polymers . the impact of the oil + surfactant / wall material weight ratio on the microparticulate properties , morphology , and size distribution was evaluated for the representative case of whey protein . the results are reported in figure 5 , and the quantitative study of the size maxima and standard deviation values are reported in figure 6 . four different ratios were tested , ie , 1:1 , 1:2 , 1:3 , and 1:4 , corresponding to vitamin e acetate loadings of 37.5 , 27.3 , 21.4 , and 17.6 wt% , respectively . the samples appear to be very homogenous , generally presenting a similar aspect . when compared with the pure formulations shown in figure 1a , the first point to observe is the almost perfect spherical shape of the particles . the deflated morphology of the particles discussed above is no longer present , now being replaced by spherical shapes and smooth surfaces . this could be due to the global increase of droplet loading before drying , which is doubled with the ratio 1:1 . hence , the empty cavities formed with pure wall materials no longer exist , giving way to spherical morphologies . as for the size distribution , a clear difference is observed between the ratios 1:1 , 1:2 , 1:3 , and 1:4 . the latter presents an outstanding submicronic size , with particle diameters around 500 nm . looking at this result in terms of interface properties , we can note that not only do the nanoemulsions here show significant surfactant properties , but that the droplet loading is reduced with the ratio of 1:4 , thus affording a decrease in the particle size . furthermore , these results were obtained with whey protein , for which each ratio gave rise to powdery and homogeneous samples . however , using the other wall materials , this limit fluctuated from 1:1 to 1:4 . wall materials were spray - dried along with surfactant alone , vitamin e acetate alone , and both the surfactant and the vitamin e acetate formulated as a nanoemulsion . for each experiment using surfactant alone and vitamin e acetate alone , the compound amounts strictly corresponded to those required for nanoemulsion formulation . the aim of these experiments was to see whether the nanodispersed forms themselves could have an influence on the spray - drying process . the results are presented in figure 7 for representative wall materials , including quantitative study of the size maxima and standard deviation values for gum arabic ( figure 7b ) , whey protein ( figure 7c ) , and kleptose and nutriose ( figure 7d ) , and are summarized in figure 8 . here , the first observation concerns the morphology of the microparticles as a function of the compounds added to the wall materials . as discussed earlier , the deflated aspect of the particles is generally inhibited by the presence of the oily phase or nanoemulsions , but , conversely , it seems to be enhanced by the surfactant ( except with the whey protein for which this deflated effect appears less pronounced ) . indeed , for a similar amount of surfactants using the wall material + surfactant alone system and the wall material + nanoemulsion system , the resulting microparticle sizes were significantly different , especially for gum arabic and whey protein . however , even if this result is not as apparent with kleptose and nutriose , the global trend highlighted in figure 8 shows that generally the size is slightly reduced with the wall material + surfactant alone system and slightly increased for the wall material + vitamin e acetate alone system . these results corroborate the idea that nanoformulated materials can present more enhanced effects than those induced by nonformulated compounds . in our case , only the surfactant is likely to affect the interface phenomena governing the spray - drying process . this can be illustrated by comparison of the wall material + surfactant alone system and the wall material + nanoemulsion system . this suggests that some of the nanoemulsion droplets are trapped at the water / air interface , reducing the surface tension even more than with the surfactant alone , and giving rise to smaller droplets . one objective of this study was to prove that it was possible to microencapsulate nanoemulsion droplets in order to propose new solutions for the delivery of nanoemulsions , thus allowing enhanced absorption of the encapsulated compounds . the microencapsulation of nanoemulsion droplets also requires that , once entrapped in the dried microparticles , the nanoemulsion oil droplets conserve their size and individual nature . the polymer is rapidly solubilized due to the huge specific surface of the powder and , if kept intact during the process , the nanoemulsion droplets can be redispersed in water . their resolubilization in water allowed redispersion of the nanoemulsion droplets . because all the wall materials used are soluble in water , the times within which the nanoemulsions are redispersed in water appear to be relatively short , ie , a few minutes . they will depend on both the water / particle ratio and the nature of the wall materials ( ie , on the kinetics of the wall material solubilization in water ) . for instance , for a given amount of powders mixed in water ( 10 wt% ) , redispersion of the nanoemulsions was achieved for the different wm as follows : gum arabic about 5 minutes ; whey protein about 5 minutes ; kleptose about 3 minutes ; and nutriose about 3 minutes . after redispersion , the samples , like typical nanometric dispersions , were translucent and their characterization was performed by dynamic light scattering . the results are reported in table 1 , presenting the hydrodynamic diameter and polydispersity index of the nanoemulsion droplets , just after formulation ( called raw nanoemulsions ) , after mixing with wall materials before and after spray - drying , and redispersion in the same volume of water . to summarize , a comparison of the samples before and after spray - drying showed a significant increase in size , and was generally doubled for all the samples . this results from slight droplet coalescence and can simply be due to turbulence and heat during the spray - drying process . the results also vary in the function of the wall materials , again indicating that the oil / polymer interactions and affinities could play a role in droplet merging . nevertheless , the average size of the redispersed droplets remained at about 150 nm and , with a polydispersity index of around 0.2 , such a suspension can still rightly be considered a nanoemulsion . taking into account all the results of the present study , a schematic illustration of the microparticulate structure is shown in figure 9 . these multiscaled systems can in fact be used as carriers for nanoemulsions , ensuring their easy manipulation , concentration , and administration . stability studies were performed on the powders , the results of which are reported in figure 10 . we can first note that , at initial time , the measurements show approximately 100% of vitamin e acetate for all the seven wall materials tested . this simply indicates that the spray - drying process does not destroy the encapsulated molecules . it is also worth noting that only one peak of vitamin e acetate was observed for all the wall materials studied and that no other peaks appeared over 3 months . in other words this result is confirmed and corroborated by the overall aspect of the curves in figure 10 , indicating a constant amount of vitamin e acetate with a recovery of approximately 100% . these results confirm that the spray - drying process can be considered an efficient method for the microencapsulation of nanoemulsions , ensuring the integrity of the molecules throughout the process , as well as their conservation during storage of the dried product . this study proposes a novel technique using a spray - drying process to microencapsulate nanoemulsions . the dried powder obtained consists of multiscaled objects in the form of microparticles , with a size range of around 2 m . the present study also demonstrates that these microparticles are able to encapsulate ( or more precisely entrap ) nanoemulsion droplets of around 150 nm in their polymeric matricial structure . the low - energy nanoemulsification method chosen , associated with the gentle form of spray - drying , makes the whole process suited to the encapsulation of sensitive molecules . the model lipophilic molecule tested was vitamin e acetate , encapsulated at around 20% in the dried powder . we show that the presence of nanoemulsion in the solutions before spray - drying can have a significant impact on the size distribution and morphology of the microparticles . however , the process itself does not destroy the oily nanodroplets , which can be easily redispersed once the powder is in contact with an aqueous phase . hplc follow - up of vitamin e acetate integrity shows that the molecules remain intact throughout the process , as well as during their conservation in dried form .

depression is a frequent yet overlooked occurrence in primary health care clinics worldwide . depression and related health screening instruments are available but are rarely used consistently . the availability of technologically based instruments in the assessments offers novel approaches for gathering , storing , and assessing data that includes self - reported symptom severity from the patients themselves as well as clinician recorded information . in a suburban primary health care clinic in quito , ecuador , we tested the feasibility and utility of computer tablet - based assessments to evaluate clinic attendees for depression symptoms with the goal of developing effective screening and monitoring tools in the primary care clinics . we assessed individuals using the 9-item patient health questionnaire , the quick inventory of depressive symptoms - self - report , the 12-item general health questionnaire , the clinical global impression severity , and a dsm - iv checklist of symptoms . we found that 20% of individuals had a phq9 of 8 or greater . there was good correlation between the symptom severity assessments . we conclude that the tablet - based phq9 is an excellent and efficient method of screening for depression in attendees at primary health care clinics and that one in five people should be assessed further for depressive illness and possible intervention .

technology based assessments are increasingly implemented for medical assessment and monitoring of individuals with a variety of medical and psychiatric disorders . weight management and diabetes monitoring programs use technological based programs effectively [ 1 , 2 ] . web - based assessments and monitoring programs have been in place to monitor psychiatric symptoms for several years [ 35 ] . telephone based systems have demonstrated the efficacy of this technology in the field worldwide [ 6 , 7 ] , including ecuador . tablet - based systems for capturing survey data are well established and used clinically as an extension of the medical data recording method . adapting technology based assessments and monitoring tools for psychiatric disorders is critical as it may improve efficiency ; responses to standardized questions generate measures for clinical monitoring that are automated and accessible . the versatility of the computer tablet allows the technology to be adapted widely in primary care clinics . however , the use of computer tablets in an emerging health system , such as in quito , ecuador , has not been studied . the feasibility at the level of the patient population and the capacity to gather and record symptoms of depression are critical to successfully adapting a technology based screening and monitoring system for psychiatric symptoms in primary care . the effective clinical management of major depression in primary care represents a significant opportunity for improvements in health , both in terms of the quality of individual life and the financial sustainability of community health delivery programs by enhanced adherence to medical interventions . depression is a common disorder and is among the prominent and leading medical causes for disability worldwide [ 10 , 11 ] . depression is recognized as a major problem in the primary health care setting with a point prevalence between 5 and 10% and up to twice as many that experience depressive symptoms but do not rise to a full major depression diagnosis . individuals with depression have a threefold increase in comorbid chronic diseases , are less likely to seek and receive effective treatment for medical conditions , have lower rates of appropriate preventative health services and screening , and are less adherent to medical recommendations . the majority of individuals with depression are treated by primary care providers who prescribe 80% of the antidepressant medications in the usa . the burden of untreated depression at the primary care level is substantial as most depressions go unrecognized and untreated . in addition to the personal and vocational cost of untreated depression , it has been shown that overall medical care costs are increased , with greater numbers of patient care provider interactions , investigations , and treatments for a variety of medical complaints . identification , diagnosis , and implementation of treatments specific for depression at the primary care level have substantial personal and medical implications for effective patient management in the community [ 17 , 18 ] . the few studies that address the prevalence of depressive disorders in ecuador are limited to the public health sector collecting data on use of health services ; by the year 2007 the rate of major depression was 72 per 100,000 inhabitants , low but a clear increase and effectively double the rate from the previous decade ; by 2012 the rate of major depression was 162 per 100,000 . compared to the usa rates , these are lower rates ; however , the increasing rates are likely to reflect an increased awareness of depression both at the care provider and patient focused levels . there is clear evidence of depressive disorders throughout the ecuadorian society , including the indigenous populations [ 21 , 22 ] . a study conducted in the rural and urban sectors of a quito health area using the ghq-12 found that 37.1% of the individuals surveyed had positive scores for mental health issues . the global health perspective on the rates of depression throughout the world suggests that the prevalence is likely to be in the 10% range , significantly higher than estimated by public health records in the ecuadorian government ( 1.6% ) and consistent with observations elsewhere that up to 75% of depressions go undiagnosed and therefore untreated . we tested the use of a computer tablet in the clinical setting to screen for depressive symptoms in a sample of attendees to a primary clinic program in quito , ecuador , in order to determine the feasibility and acceptance of the implementation of novel technology to assess depression symptoms by the patient population and compare two depressive symptom severity instruments , the 9-item patient health questionnaire ( phq9 ) and the quick inventory of depressive symptoms - self report ( qids - sr ) , with the 12-item general health questionnaire ( ghq-12 ) . we find that the technology was readily embraced and we find identified rates of depression in the attendees of a primary care clinic that were similar to those in the usa . we hypothesized that the clinic attendees would embrace the novel technology and that the screening questionnaires would identify individuals with depressive symptoms by increasing the efficiency of the clinical interaction . patients attending a primary health care clinic affiliated with universidad san francisco de quito in quito , ecuador , were invited to participate anonymously in a series of self - administered questionnaires . individuals were offered participation by the clinic staff at arrival and were evaluated prior to seeing the primary care physician ( pcp ) . after informed consent was procured and the participants completed a short tutorial on the use of tablet ( ipad ) in completing the questionnaires embedded in the polldaddy survey software application , participants were asked to independently complete all three screening tools . the patients were screened by a team of medical students ( ds , ef , and gj ) for depressive symptoms using a dsm - iv checklist for major depressive disorder by the clinical researchers . finally , the clinical global impression - severity ( cgi - s ) rating by the primary health care provider recorded the clinician 's impression of patient 's level of depression from 1 ( not at all depressed ) to 7 ( critically depressed ) . the cgi - s was completed after the survey questions were completed and after the individual had seen the pcp . the instruments used were the nine - item patient health questionnaire ( phq-9 ) [ 29 , 30 ] , ghq-12 ( general health questionnaire ) [ 27 , 31 ] , and qids - sr ( quick inventory of depressive symptomology - self report ) [ 26 , 32 ] . the spanish language versions of assessment instruments were uploaded to a central website ( http://www.polldaddy.com ) designed to host survey questionnaires that could be administered in the field using a computer tablet ( ipad ) . the qids - sr was scored according to the method described by the epidemiology data center of the university of pittsburg ( http://www.ids-qids.org/index2.html#scoring ) . reliability was evaluated by comparing scores on the phq-9 with those on a measure reflecting depression symptomology previously studied in latin american patients , the ghq-12 . convergent and divergent validity was assessed with spearman 's rho , comparing phq-9 scores to the scores of the ghq-12 and qids - sr . feasibility was assessed quantitatively by recording the proportion of patients that were able to complete the questionnaires once they had agreed to participate . it was also qualitatively assessed through observations from the research assistants regarding ease of use among the different demographics that participated in the study . this study engaged 226 participants between the ages of 18 and 65 , attending a primary care clinic for nonpsychiatric reasons , that agreed to participate anonymously in a survey of depressive symptomatology using computer tablet - based technology . all 226 patients completed the phq-9 , the ghq-12 , and the qids - sr . based on the average number of daily contacts at the clinic , it is estimated that there were approximately 500600 individuals that attended the clinic during the study time . a total of 131 cgi scores for these patients were collected as well ; this proved to be somewhat more difficult to gather as it required the clinical researcher to interact with the primary care clinician , whose availability was often limited . the dsm - iv major depressive episode checklist review was conducted on 188 patients ( table 1 ) . table 2 presents the correlation coefficients between the phq-9 and the other depression assessment tools . the phq-9 results were compared to the data from the ghq-12 , the qids - sr , the dsm - iv , and the cgi . the phq-9 showed significant correlation with all measures : ghq-12 ( r = 0.64 , p < 0.0001 ) , qids - sr ( r = 0.68 , p < 0.0001 ) , dsm - iv symptom count ( r = 0.67 , p < 0.0001 ) , and cgi ( r = 0.39 , p < 0.0001 ) . figure 1 further demonstrates the relationship between the phq measures with the qids - sr , ghq-12 , and cgi . despite the apparent correlation , there was a broad range of cgi scores and 2/3 of individuals with cgi of 3 or less had a phq9 > 8 , with the implication of depressive symptoms not suspected clinically . all participants who started the survey ( 226/226 ) were able to complete the self - assessments on the ipad tablet computer . five patients were unable to read the text on the tablet screen as a result of not bringing reading glasses to the doctor 's office and were therefore excluded from participating . the integration of technology in the assessment and monitoring of depression represents a tangible advance in clinical care and increases efficiency in detection and treatment , particularly in communities with limited resources and capacity for medical care ( device costs are decreasing ) . screening for depression in a primary care setting of quito , ecuador , using computer tablet - based technology proved to be a very efficient and adaptable method of gathering information ; it was readily accepted by the clinic attendees and used without difficulty following a brief orientation to the process by the researchers . a phq9 score of 8 was used as a threshold for screening purposes ; it is well known that somatic symptoms are common ( 75% ) in latin american culture and there is a tendency to under represent mood symptoms . in many primary care clinics , 75% of patients subsequently found to be depressed initiated the clinical visit because of somatic concerns , and depression was not the self - identified driving need for medical attention . this argues for a lower threshold on the phq9 in cultures wherein psychological aspects of depression are likely to be less frequently acknowledged ; we believe this will increase the sensitivity of the screening . there were instances initially wherein presbyopic participants were challenged and did not bring along reading glasses ; this was solved by having off - the - shelf corrective reading glasses available ; 4 patients who were unable to complete the forms are not included in the total 226 participants . all self - report questionnaires had been applied to psychiatric research and the construct validity of the measures has been established in the usa clinical setting [ 33 , 36 , 37 ] . in addition to piloting the use of technology at the clinical primary care level of quito , it was our intent to evaluate the depressive symptom screening and severity instruments to estimate their clinical utility . we considered a control group with paper based screening ; however , the project was designed to assess how well a technological approach could be adapted to an active clinic . we elected to use participation and completion of assessments as a measure of acceptability ; in view of the fact that we were integrating into the clinic process and wished to gather data in an expedited manner , we elected to not gather information on the perspective of the participant on the process . while this may be considered a weakness of the study , we feel that the strength of the approach is the assumption that measurement based care is integral to health care at the primary level . there was excellent correlation between the depression symptom severity assessment scores of the phq-9 and the qids - sr ( 0.68 ) which is expected as they are both designed to measure depression ; however , this correlation is important as we are in fact measuring whether depression is identified with 2 separate measurement instruments . there was excellent correlation with the number of dsm - iv symptoms acknowledged by the participant and their phq-9 scores , and , finally , there was good correlation with the ghq scores , consistent with previous findings . studies of the phq9 in latin america are few ; however , a study in honduras indicated that it was a feasible measure of depression in this culture . the ghq has been validated in chile as an efficacious measure of depressive symptomology , and has been used in ecuador . thus , we conclude based on our correlated results that are consistent with strong convergent validity of the measures that they are valid measures of depressive symptoms in the ecuadorian population . the clinical global impression - severity ( cgi - s ) scale is a 7-point scale that is used to record the clinician 's impression of the severity of the patient 's illness at the time of assessment . it is a subjective scale and is generally reflective of the knowledge of the clinician about the patient and their illness . the correlation of cgi - s with depressive symptom ratings is generally good and can be expected to predict symptomatic states . the observation that a substantial number of individuals with a cgi - s of > 3 were present despite having relatively substantial symptoms of depression suggests that the clinicians who were asked to give their impression of the patient 's depressive symptoms were unaware of the depression . this could be for a number of reasons ; the patient may have attended the clinic for reasons not related to how they were feeling and did not disclose to the primary care physician that they were depressed . the pcp may not have asked about depressive symptoms and was unable to determine therefore if depression was present . the pcp may not have been attuned to the patient 's medical complaints and was not concerned that the physical complaints could be driven in part by underlying depressive symptoms . the health care system in ecuador is state funded through the ministry of health ; the first line of contact with the health care system is through the primary care physician who is responsible for the implementation of the government 's health care policies . ongoing efforts are focused on strategic implementation of preventative health screening in several dimensions such as diabetes and hypertension , but currently there are no policies for screening for depression or other psychiatric disorders . there is every advantage to a national screening program implemented systematically through the primary health care providers . there is considerable efficiency in screening for depression in the primary care setting [ 41 , 42 ] , particularly when available care is at hand . on the other hand , if there are no specific interventions to follow up on the screening process , there is questionable utility in screening at the population or primary care level for depression . since the management of depression is done by primary care providers in most health systems , it is most logical that the ecuadorian health system engage their pcps in the screening and management of depression . the paucity of published research in depression in the latin american countries implies that they have similar challenges as ecuador . there are several advantages to screening depression and the use of electronic methods provides efficiency in data collection and management . the instruments used in the current study are easily adapted to electronic versions and showed high correlation between scores which suggests a consistency of measure of the condition ( depression ) that they were designed to assess . this is of importance at the level of primary care , where there frequently is no psychiatrist or mental health professional available to perform clinical evaluations . the importance of screening is reflected in the next steps , specifically what happens with the information and the subsequent actions . at what severity level we find that 20% of individuals have a phq-9 > 8 and would recommend further evaluation at the primary care level for major depression and possible treatment . given the level of comorbid medical chronicity and other potential personal , medical , and social consequences of depression , the combined economical and health benefits of treatment of depression are clear . there is evidence that medical assistants are effective in follow - up of screening assessments and facilitators of treatment and intervention strategies , providing practical assessment to the primary care team before the referral for specialty evaluation . the strategies of an electronically based screening process such as that performed in this project are an effective first step in the identification and management of depression . the limitations of this report include the lack of a thorough follow - up examination with a detailed clinical interview to verify the diagnosis . further , this was not a systematic screening of attendees and so a rate of depression can not be established . the primary purpose of this work was to assess the efficiency of the tablet - based screening in the clinics . we find that one in five individuals attending a primary health care clinic reports a phq-9 > 8 and of those individuals only a small percentage had cgi - s scores > 3 suggesting that concerns were noted by the clinician infrequently . the cut - off of 810 for the phq-9 has been suggested in populations with somatic complaints . this is consistent with the observation in the literature that approximately 50% of depressions are missed in primary care . we were able to gather cgi - s in only half of our participants , as the availability of the pcp was limited , which again reflects the busy nature of a primary health care clinic . depression is frequently overlooked in the clinical setting in ecuador and this is reflected in the low rates of depression reported by the ministry of public health . the recent increases in the reported rates of depression suggest that there is an increasing awareness to this disease ; however , the rates are still substantially lower than elsewhere and there is every likelihood that the ecuadorian rates parallel those elsewhere . primary care is clearly the clinical contact point for individuals to be screened for depression and use of computer technology in the screening process is demonstrated to be very effective and acceptable to the population . the phq-9 and the shorter phq-2 are excellent methods for screening for depressive symptoms to be followed up with a more detailed assessment and treatment . screening for depression is only one step in the overall management of depression ; clearly it is necessary to detect and diagnose the illness in order to effectively treat it . however , merely detecting depression at the primary care level in the absence of a program to manage the disorder is of minimal consequence [ 46 , 47 ] . detecting elevated blood glucose is of no use unless a system is in place to diagnose and treat diabetes . they range from a diagnostic interview by a clinician with experience in mental health to determine the nature of the problems and the specific needs of the patient to education programs on health and illness management . the treatment plan is subsequently tailored to the needs of the patient and the capacity of the care providers . critical to the management is a systematic method of screening and monitoring of symptoms and we have demonstrated that a tablet - based system is an effective and acceptable method that is easy to use in any clinic setting .

although candidiasis in newborns is not uncommon , superficial dermatophyte infections of infants is quite rare . the causative agents of neonatal tinea reported in various case studies have been trichophyton rubrum , microsporum canis , microsporum gypseum , and trichophyton violaceum . to the best of our knowledge , no case report of neonatal tinea faciei caused by trichophyton mentagrophytes has been reported earlier .

it is often a deceptive facial eruption , and can mimic a variety of cutaneous dermatoses . the pattern of infection depends on the geographic location or the endemic dermatophyte strains of a given area or the cultural population habits . tinea faciei infections are common in children but they are rare in infants . although neonatal tinea is rare , cases have been reported occasionally . a 20-day - old female child presented with multiple , annular lesions over the face for last 4 days . on examination , erythematous annular plaques of size 23 cm in diameter were present over the face ( both cheeks , forehead ) with well - defined irregular raised margins [ figure 1 ] . the child was apparently normal at birth with uneventful labor and normal vaginal delivery and was breastfed . on enquiring , the mother gave a history of itching and extensive annular erythematous plaques over both axilla , inframammary area , abdomen , both thighs , inguinal folds , buttocks since her first month of pregnancy [ figure 2 ] . she was given only topical treatment to avoid any side effects of drugs in pregnancy . neonatal tinea faciei ; ( left ) annular lesion on both cheeks with active periphery , ( top right ) right cheek and forehead , and ( bottom right ) left cheek tinea corporis in mother , ( left ) inframammary area , ( top right ) buttocks , and ( bottom right ) inguinal folds in the child , potassium hydroxide ( koh ) examination of skin scrapings from the active edge revealed the presence of numerous thin , long , septate , branched hyaline hyphae [ figure 3 ] . culture on sabouraud 's dextrose agar grew white granular to powdery colonies after 10 days of incubation [ figure 4 ] . koh examination of skin scrapings showing branched hyphae creamy white powdery colonies of trichophyton mentagrophytes on sabouraud 's dextrose agar lcb mount revealing abundant microconidia in clusters the mother was treated with oral terbinafine 250 mg and topical luliconazole for 4 weeks , and the neonate responded within 7 days to topical clotrimazole alone without any clinical adverse effects [ figure 6 ] . healed lesions of neonatal tinea faciei after 7 days by definition , all dermatophyte infections of face in women and prepubertal boys are tinea faciei . tinea faciei is often misdiagnosed as seborrhoeic dermatitis , atopic dermatitis , bacterial infections , irritant contact dermatitis , cutaneous lupus erythematosus , rosacea , granuloma annulare , perioral dermatitis , pityriasis alba , and pityriasis rosacea . the clinical manifestations are mainly attributed to the immune response of the host to the invading fungal species . although antigen presentation in neonates is intact , t - cell function is still inadequate to mount an appropriate immune response against fungal infections . the rare presentation of neonatal tinea faciei is probably explained by the immaturity of their immunological system and also because dermatophytes require an incubation period of 13 weeks to produce clinical manifestations . atypical features are more common in tinea faciei than in other forms of ringworm infections because of the complex anatomy of the face . the variable expression can be attributed to the degree of inflammation and depth of invasion . at the onset of disease , clinical features vary considerably , but it is often associated with burning , itching , and photosensitivity . mycological examination is vital in arriving at a confirmatory diagnosis , especially in cases with variable morphology . smears are labeled as provisionally positive when branched , translucent , nonpigmented , thin , and septate mycelia are seen . culture on sabouraud agar is done for 34 weeks for the presence of characteristic fungal colonies . microscopic and culture examination in this neonate was positive for t. mentagrophytes , both for the mother and the child . previous case reports of neonatal tinea faciei have reported trichophyton rubrum , microsporum canis , and microsporum gypseum . ghorpade et al . have reported t. mentagrophytes in tinea corporis in a 2-day - old neonate , but to the best of our knowledge , tinea faciei in a 20-day - old neonate caused by t. mentagrophytes has not been reported earlier . the neonatal tinea faciei in our patient shared the same pathogenic fungus causing concurrent tinea corporis with her mother , illustrating the epidemiological association between close contact and infection . the contact between the baby 's face and her mother 's breast during breast feeding may be the main reason of transmission and infection of the face . same species has been reported previously by ghorpade et al . to cause tinea corporis in a 2-day - old neonate . controversy remains on whether topical or systemic antifungals should be used to treat dermatophytoses in neonates . ironically , topical antifungals are proposed to work more effectively in infants due to the same risk factor that predisposes them to tinea infection , that is , increased permeability due to immature epidermis . although terbinafine should be carefully used in pregnancy and lactation , it can be considered for extensive tinea corporis infections as it is a category b drug . mother had extensive tinea corporis ( both axilla , inframammary area , abdomen , both thighs , inguinal folds , buttocks ) from the onset of first month of pregnancy , and had taken only topical antifungal combinations during pregnancy with partial relief . she had previous history of partially treated tinea corporis over 5 years , and on grounds of the extensive area of the disease , severe itching and her rural background , she was given oral antifungals . neonatal tinea is rare , therefore its occurrence gives a clue to the dermatophyte infection occurring due to intimate contacts , hence complete examination of the source and treatment of infected pets should be carried out to control the infection . although the person to person transmission route is most likely , the role of environmental reservoirs such as bed linen , mattresses should not be ignored . although neonatal tinea corporis due to t. mentagrophytes has been reported in the past , what makes our case interesting is the fact that it has never been reported to cause neonatal faciei earlier . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed .

objective : to evaluate the melatonin levels in saliva in gingivitis and periodontitis cases.background:melatonin has strong antioxidant , free radical scavenging , and immunomodulating properties , acts on osteoblasts directly to stimulate cell proliferation and synthesis of type i collagen , and promotes bone formation.materials and methods : a total of thirty participants were selected and divided into three groups ( control group , gingivitis group , and periodontitis group ) . in each group , ten participants were taken . salivary melatonin was estimated in each of the three groups.results:results from this study showed that the mean levels of salivary melatonin increased as severity increased from control to periodontitis , i.e. , the mean levels were highest in periodontitis followed by gingivitis and least in control group . the melatonin level of all participants was positively and significantly ( p < 0.01 ) correlated with their gingival index ( r = 0.85 , p < 0.01 ) and probing depth ( r = 0.72 , p < 0.01).conclusion : salivary melatonin level varied with the severity of gingivitis and periodontitis . with increased severity of periodontal disease , the level of salivary melatonin also increased suggesting that salivary melatonin may act as a diagnostic biomarker for periodontal diseases .

the status of disease ranges from gingivitis to advanced periodontitis with destruction of connective tissue attachment and alveolar bone which can ultimately lead to tooth loss . periodontal tissue damage is caused by direct effect of the toxic products released by the bacterial plaque and from the action of the host immune system , stimulated by the bacterial infection . the important characteristic of periodontal disease pathogenesis is the generation of free radicals , some of which are derived from the bacteria themselves and others are derived from the host immune response . it has been suggested that increase in both reactive oxygen species ( ros ) which are hydroxyl ( oh ) radical , hydrogen peroxide ( h2o2 ) , singlet oxygen ( o2 ) , and hypochlorous acid ( hocl ) and reactive nitrogen species which are nitric oxide , peroxynitrite anion ( onoo ) , and peroxynitrous acid ( onooh ) during periodontitis is responsible for the oxidative damage to periodontal tissues . this disturbed balance between the pro - oxidant and antioxidant systems may result in added oxidative attack and extensive worsening of the periodontal tissues . it affects many physiologic processes , such as the control of circadian rhythm , the regulation of the body temperature , and the activation of the immune system . after the release of melatonin in blood stream the fraction of plasma melatonin passing through salivary glands into the mouth appears to be constant ranging from 24% to 33% . about 70% of plasma melatonin this study was conducted with the aims and objectives to observe the melatonin levels in saliva in gingivitis and periodontitis cases . a total of thirty participants with age group between 18 and 65 years were selected for this study from the department of periodontics , faculty of dental sciences , king george 's medical university , lucknow , uttar pradesh , india . ten participants each were taken from control group ( without disease ) , gingivitis group , and periodontitis group on the basis of disease severity . participants without the presence of diseases which may affect the immune system ( such as chronic infections and oncological disorders ) and treatment which can change the melatonin levels were enrolled . before starting the study , informed written consent from all the participants was obtained . all the participants selected for this study underwent oral examination for periodontal assessments , and medical history was taken . gingival index by loe and silnes method and probing depth measured by unc-15 probe were recorded for each participant . beverages containing artificial colorants as well as alcohol , coffee , and bananas were avoided during sampling period . salivary flow stimulation by chewing gums and eating lemons was also avoided to prevent any possible interference . cotton swab provided in the device was kept in the patient 's mouth below the tongue . frozen samples were thawed and centrifuged at 1000 g and 2c8c for 5 min before assay . the amount of melatonin in saliva was determined by the buhlmann direct saliva melatonin elisa , a competitive immunoassay using a capture antibody ( ab ) technique , according to manufacturer 's instructions . after the first 1620 h overnight incubation , melatonin present in cases and controls as well as in the calibrators competed with biotinylated melatonin during the second 3-h incubation for the binding sites of this highly specific ab . after washing , the enzyme label , streptavidin , conjugated to horseradish peroxidase is added , this binds during the third incubation step ( 1 h ) to the melatonin - biotin ab complexes captured on the coated wells . the unbound enzyme label was then removed by the second washing step , and tetramethylbenzidine substrate was added to the wells . in the fourth 30-min incubation step , a chromophore was formed in inverse proportion to the amount of melatonin present in the sample . the concentration of melatonin was determined in picograms / milliliter ( pg / ml ) . due to small sample size , heterogeneous variance , and skewed nature of the data , wallis ( h ) one - way analysis of variance ( anova ) , and the multiple comparisons of mean ranks between groups were done by z - test . spearman rank - order correlation ( rs ) method was used to assess relative association among the variables . a two - tailed ( = 2 ) p < 0.05 was considered to be statistically significant , p < 0.01 as highly significant , and p > 0.05 the not significant . beverages containing artificial colorants as well as alcohol , coffee , and bananas were avoided during sampling period . salivary flow stimulation by chewing gums and eating lemons was also avoided to prevent any possible interference . cotton swab provided in the device was kept in the patient 's mouth below the tongue . frozen samples were thawed and centrifuged at 1000 g and 2c8c for 5 min before assay . the amount of melatonin in saliva was determined by the buhlmann direct saliva melatonin elisa , a competitive immunoassay using a capture antibody ( ab ) technique , according to manufacturer 's instructions . after the first 1620 h overnight incubation , melatonin present in cases and controls as well as in the calibrators competed with biotinylated melatonin during the second 3-h incubation for the binding sites of this highly specific ab . after washing , the enzyme label , streptavidin , conjugated to horseradish peroxidase is added , this binds during the third incubation step ( 1 h ) to the melatonin - biotin ab complexes captured on the coated wells . the unbound enzyme label was then removed by the second washing step , and tetramethylbenzidine substrate was added to the wells . in the fourth 30-min incubation step , a chromophore was formed in inverse proportion to the amount of melatonin present in the sample . the concentration of melatonin was determined in picograms / milliliter ( pg / ml ) . due to small sample size , heterogeneous variance , and skewed nature of the data , the studied variables were analyzed using nonparametric tests . wallis ( h ) one - way analysis of variance ( anova ) , and the multiple comparisons of mean ranks between groups were done by z - test . spearman rank - order correlation ( rs ) method was used to assess relative association among the variables . a two - tailed ( = 2 ) p < 0.05 was considered to be statistically significant , p < 0.01 as highly significant , and p > 0.05 the not significant . the salivary melatonin level , gingival index , and probing depth of three groups of participants were shown in graphs 1 and 2 and summarized in tables 1 and 2 , and clinically correlating photographs are given in figures 13 . the salivary melatonin levels of control group ranged from 0.80 pg / ml to 6.80 pg / ml with an average ( standard error [ se ] ) 3.92 0.58 pg / ml while it ranged from 3.30 pg / ml to 10.90 pg / ml with an average ( se ) 6.87 0.82 pg / ml in gingivitis group and from 5.20 pg / ml to 14.80 pg / ml with an average ( se ) 10.20 0.98 pg / ml in periodontitis group . mean salivary melatonin levels ( pg / ml ) in participants of control group , gingivitis group , and periodontitis group box and whiskers plot showing the distribution of melatonin level ( pg / ml ) , gingival index , and probing depth ( mm ) in three groups of participants summary of melatonin level ( pg / ml ) of control group , gingivitis group , and periodontitis group participants one - way analysis of variance of salivary melatonin levels of control group , gingivitis group , and periodontitis group participants clinical photograph of control group participant in which salivary melatonin levels ( pg / ml ) were lesser than other two groups ( mean = 3.92 ) clinical photograph of gingivitis group participant in which salivary melatonin levels ( pg / ml ) were greater than control group and lesser than periodontitis group ( mean = 6.87 ) clinical photograph of periodontitis group in which highest levels of salivary melatonin ( pg / ml ) were found ( mean = 10.20 ) comparing the levels of salivary melatonin of all three groups together , the anova [ table 2 ] revealed that the mean levels of salivary melatonin among groups differed significantly ( f = 14.93 , p < 0.01 ) . the correlation between severity ( severity of disease of control group was ranked 1 , gingivitis as 2 , and periodontitis as 3 ) , melatonin level , gingival index , and probing depth in all three groups of participants was summarized in table 3 . correlation ( n=30 ) between variables of three groups of participants estimation of melatonin level from each gingival index and probing depth was also done separately by simple linear regression analysis , considering the melatonin level as a dependent variable and gingival index and probing depth the independent variables [ graph 3 ] . the regression analysis showed significant effect of both gingival index ( f = 33.24 ; p < 0.01 ) and probing depth ( f = 23.35 ; p < 0.01 ) on melatonin level which can account ( estimate ) 54.3% and 45.5% of the total variations ( i.e. , coefficient of determination ; r ) of melatonin level , respectively . the best fit regression equation to estimate melatonin level from gingival index and probing depth follows as : melatonin level ( pg / ml ) = 3.37 + 2.79 gingival index ( score ) ( i)melatonin level ( pg / ml ) = 0.92 + 2.10 probing depth ( mm ) . ( ii ) melatonin level ( pg / ml ) = 3.37 + 2.79 gingival index ( score ) ( i ) melatonin level ( pg / ml ) = 0.92 + 2.10 probing depth ( mm ) . ( ii ) best fit regression equation to estimate melatonin level ( pg / ml ) from gingival index ( scores ) ( a ) and probing depth ( mm ) ( b ) with 95% confidence interval for ( broken lines ) periodontal diseases are known to be exacerbated by free radicals and by altered host immune response to microorganisms present in plaque . in periodontitis , in addition , a considerable migration of neutrophil to the gingiva and gingival fluid during periodontitis leads to excessive production of ros . hence , this increase in the free radical production in periodontal diseases can lead to rise in melatonin level due to its free radical scavenging and antioxidant properties . melatonin can alter the development of the periodontal disease because it can act on pge2 , thereby inhibiting the differentiation of the osteoclasts induced by cell - to - cell contact between osteoclasts and osteoblasts . melatonin can also modulate all proteins that regulate the resorption process in the periodontal disease and interact with other biologic agents such as calcitonin , parathormone and 1 25(oh)2d3 , or interleukin ( il)-2 , il-1 , and il-6 , and osteoprotegerin / receptor activator of nuclear factor-b ligand system . in contrast to previous studies which reported decreased salivary and serum melatonin levels in periodontitis , in the present study , authors reported increased salivary melatonin levels with increased severity of periodontal disease and suggested that this increase may be result of a signal(s ) derived from periodontal inflammation in oral cavity . further research is required to make clear the role of melatonin in the pathogenesis of periodontal disease and to understand its clinical relevance .

objectivethe aim of this study was to evaluate the marginal adaptation of biodentine in comparison with mineral trioxide aggregate ( mta ) and intermediate restorative material ( irm ) , as a root end filling material , using scanning electron microscopy ( sem).materials and methods : thirty permanent maxillary central incisors were chemo - mechanically prepared and obturated . three millimetres of the root end were resected and 3 mm retro cavity preparation was done using ultrasonic retrotips . the samples were randomly divided into three groups ( n=10 ) and were restored with root end filling materials : group i mta , group ii biodentine , group iii irm . the root ends were sectioned transversely at 1 mm and 2 mm levels and evaluated for marginal adaptation using sem . the gap between dentin and retro filling material was measured at four quadrants . the mean gap at 1 mm level and 2 mm level from the resected root tip and combined mean were calculated . the data were statistically analyzed , using one - way anova and tukey s hsd post hoc test for intergroup analysis and paired t - test for intragroup analysis.results:the overall results showed no statistically significant difference between mta and irm but both were superior when compared to biodentine . at 1 mm level there was no statistically significant difference among any of the tested materials . at 2 mm level mta was superior to both irm and biodentine.conclusion:in overall comparison , mta and irm were significantly superior when compared to biodentine in terms of marginal adaptation , when used as retrograde filling material .

the success of periapical surgery is dictated by elimination of infected tissues and adequate apical seal . ideal apical seal prevents ingress of residual irritants into the periapical region and percolation of periapical tissue fluid in to the canal system . various root end filling materials have been tested for their sealing ability and newer materials are still under research . the root end filling material should possess ideal properties such as bio - compatibility , dimensional stability , radiopacity , ability to set in a wet environment , antibacterial properties , easy handling , adequate compressive strength and hardness , osteoinductive and osteoconductive properties and adherence to the canal walls to provide a good apical seal . among the various root end filling materials tested , mineral trioxide aggregate ( mta ) has shown good sealing ability and biocompatibility in previous in - vitro and in - vivo studies . in recent years , various materials like biodentine , cer ( cemento endodontico rapido / fast endodontic cement ) , errm ( endosequence root repair material ) and endocem ( mta - derived pozzolan cement ) have been introduced with the aim of overcoming some of the disadvantages of the mta , such as the difficulties in handling and long setting time [ 10 , 11 , 12 ] . biodentine powder is mainly composed of highly pure tricalcium silicate , which regulates the setting reaction . the liquid contains calcium chloride ( setting accelerator ) , water reducing agent ( super - plasticizer ) and water . the manufacturer claims that this material can be used for pulp capping , pulpotomy , apexification , root perforation , internal and external resorption and also as a root end filling material in periapical surgery . in the previous studies , biodentine showed biocompatibility and the ability to induce odontoblast differentiation and mineralization in cultured pulp cells . the main benefits of biodentine over other calcium silicate based materials are the reduced setting time , better handling and mechanical properties . the importance of marginal adaptation is that it may have an indirect correlation with the sealing ability of retro - filling materials . there is no previous study assessing the marginal adaptation of biodentine when used as a root end filling material . hence , the aim of this study was to evaluate the marginal adaptation of biodentine in comparison with mta and irm , as a root end filling material , using scanning electron microscopy ( sem ) . all the teeth were cleaned , autoclaved and stored in 0.2% thymol solution until they were used . access cavity preparation was done using a # 2 round diamond point ( nsk , japan ) and coronal preflaring was done using gates - glidden drills ( mani , inc , japan ) . size # 10 k - file ( mani , inc , japan ) was introduced into the root canal until it was visible at the apex and then 1 mm was subtracted from that point to establish the working length . biomechanical preparation was done using step - back technique with apical enlargement up to # 60 size k - file ( mani inc . , japan ) . copious irrigation with 3% sodium hypochlorite ( vensons , india ) was done all through the procedure . final irrigation was done with 17% edta ( prime dental products , india ) followed by 3% sodium hypochlorite for 1 minute each and rinsing with saline . the canals were dried using absorbent points and obturation was done with 2% gutta percha points ( dentsply maillefer , china ) and zinc oxide eugenol sealer ( bombay burmah trading corp . , mumbai , india ) , using the lateral condensation technique . after 24 hrs . of obturation , the root ends were resected 3 mm from the apex using a no.1557 fissure bur ; retrograde cavity was prepared to a depth of 3 mm coaxially using surgical ultrasonic retro - preparation tips ( satelec as6d , france ) . then the teeth were randomly divided into 3 groups , with each group containing 10 teeth . group 1- mta ( dentsply / tulsa dental , tulsa , ok ) , group 2- biodentine ( septodont , saint maurdes fosss , france ) , group 3- irm ( dentsply international inc , u.s.a ) . all root end filling materials were placed incrementally , following which radiographs were taken labio - palatally and mesio - distally to confirm proper filling of the material . after root end filling , a moist cotton pellet was placed on mta for setting of the material and all the samples were stored in relative humidity ( 95% ) at 37c for 5 days . the samples were mounted in a resin block to create a platform for sectioning with a hard tissue microtome and sectioned apically at 1 mm and 2 mm levels from the apex . the samples were gold sputtered and viewed under scanning electron microscopy ( 1000x magnification ) for evaluating the adaptation of the material to the canal walls . the largest gap present between the material and canal wall was measured in four quadrants ( fig 1 and fig 2 ) and the mean gap was calculated for each sample . one - way anova and tukey s hsd post - hoc test were done for intergroup analysis to compare the 1 mm , 2 mm and overall values of the three groups ; paired t - test was used for intragroup analysis to compare the 1 mm and 2 mm values within each group , with the significance level of 0.05 using spss version 17.0 software . one - way anova and tukey s hsd post - hoc test were done for intergroup analysis to compare the 1 mm , 2 mm and overall values of the three groups ; paired t - test was used for intragroup analysis to compare the 1 mm and 2 mm values within each group , with the significance level of 0.05 using spss version 17.0 software . the overall results showed that the mean gap at the dentin - retrograde filling material interface was maximum for biodentine ( 1 . intra - group and inter - group comparison at 1 mm level and 2 mm level are shown in table 1 . at 1 mm level a successful periapical surgery requires appropriate root - end resection , preparation and adequate apical seal . in root - end resection at least 3 mm of the root - end must be eliminated to reduce 98% of the apical ramifications and 93% of the lateral canals , which might be responsible for endodontic failure . hence 3 mm of root - end was resected perpendicular to the long axis of the tooth in this study . ultrasonic tips were reported to have better control and ability to stay centered in the canal and reduce perforation risk . since diamond coated ultrasonic tips reduce the chance for microcrack formation , the diamond coated ultrasonic tips were used to prepare a 3 mm retrograde cavity coaxially . incremental placement of the retrograde filling material was done to minimize the voids and enhance the quality of the filling . numerous materials are used as retrograde filling material namely mta , gic , irm , super eba and composite resins . biodentine is a relatively new tricalcium silicate based material , which forms hydrated calcium silicate gel ( csh ) and calcium hydroxide on hydration . sem aids in assessing the marginal adaptation at the filling material - tooth interface under higher magnification . claimed that the longitudinal type of sectioning might create false gaps in the interface between dentin and root end filling material thereby affecting the evaluation of marginal adaptation . a few previous studies have evaluated the marginal adaptation of the root end filling material to the canal wall using resin replicas . orosco et al . stated that for evaluation of marginal adaptation of the retrofilling material , the samples can be directly viewed under sem after gold sputtering and there is no need for creation of resin replicas , as direct sem evaluation of the samples did not result in artificial gap formation . marginal adaptation was evaluated only at 1 mm and 2 mm levels ; 3 mm level section was avoided , as it would encroach upon the junction between the retrograde filling material and the gutta - percha . it is not clear why at 1 mm level there was no difference in marginal adaptation among the three tested materials . but at 2 mm level , mta was superior to irm and biodentine and this superiority of mta over irm for marginal adaptation is in accordance with a previous report by torabinejad et al . in a recent study , a fast setting mta - derived pozzolan cement ( endocem ) , showed tight sealing with the mould comparable to mta . but in this study , biodentine which is a fast setting tricalcium silicate based material showed inferior marginal adaptation when compared to mta and irm . in previous clinical trials , comparable surgical success rates were reported for mta and irm . in a randomized controlled trial on surgical success rate , showed that mta and irm had similar clinical results when used as a root end filling material . another clinical study by zuolo et al . showed that the surgical success rate of irm as a root end filling material was 91.2% clinically and radiographically , for a follow up period of 4 years . the inadequacy in the marginal adaptation may influence the sealing ability and the clinical success rate . a few properties such as biocompatibility and the ability to induce mineralization have been studied earlier . these properties need to be investigated , since biodentine has a very limited published literature supporting its use . as far as this in - vitro study can discern , it may be concluded that : marginal adaptation at the 1 mm level was similar among mta , irm and biodentine . at the 2 mm level , mta was superior to both irm and biodentine . in the overall comparison ,

a 14-year - old boy underwent emergency debridement surgery of right foot under spinal anaesthesia . four hours after the surgery , the patient developed symptoms of cauda equina syndrome ( ces ) . postoperative magnetic resonance imaging of the patient 's spine suggested underlying tubercular arachnoiditis . the boy was started on intravenous methylprednisolone and antitubercular therapy . he responded to the therapy and recovered completely in 2 weeks without any residual neurological deficits . we suggest that underlying pathological changes in the subarachnoid space due to tubercular arachnoiditis contributed to maldistribution of the local anaesthetic drug leading to ces .

we report a case of cauda equina syndrome ( ces ) after a spinal anaesthesia in a patient with underlying asymptomatic tubercular arachnoiditis . in this case , this disease pathology may have contributed to maldistribution of intrathecally administered local anaesthetic leading to ces . after informed consent of the patient 's parents , the resident anaesthesiologist elected to give him spinal anaesthesia for the surgery . with the routine monitors attached to the patient in sitting position , lumbar puncture was done in a single attempt with a 25 g spinal needle in l3 - 4 interspace . there was no bloody tap and the cerebrospinal fluid ( csf ) drawn was clear and colourless . thereafter , 50 mg of 5% hyperbaric lignocaine was injected in subarachnoid space after barbotage ( dilution to 2% concentration and a total volume of 2.5 ml ) . paresthesias or pain at the time of lumbar puncture or injection of the drug was absent . the surgery lasting for 45 minutes was completed uneventfully with all the haemodynamic parameters remaining stable . the patient was then shifted to recovery room . after four hours in the recovery , there had been no regression in his sensory block , which had remained at t10 . similarly there had been no recovery of the motor power in the lower limbs and deep tendon reflexes were absent . patient 's clinical features were suggestive of ces , he was started on intravenous methylprednisolone ( 500 mg / day ) and shifted to intensive care unit ( icu ) for further management . a neurologist 's opinion was sought who confirmed the finding of ces and advised for an urgent magnetic resonance imaging ( mri ) of the spinal cord . the mri imaging of lumbosacral region of spinal cord revealed arachnoiditis with dural ectasia , myelitis with intradural granulomas , clumping of cauda equina nerve roots and fluid collection close to the right posterior paraspinal muscle [ figure 1 ] . as the mri findings were indicative of tubercular arachnoiditis , antitubercular drugs ( isoniazid , pyrizinamide , ethambutol , rifampicin and pyridoxine ) were started while continuing intravenous methylprednisolone ( 500 mg / day ) . a serum sample for polymerase chain reaction ( pcr ) test for tuberculosis was sent which was later reported positive . sagittal t2-weighted mr image of dorsolumbar spine shows csf loculation ( asterix ) and clumped cauda equine nerve roots ( arrow ) in the icu , on postoperative day 2 the patient showed gradual recovery of sensations in lower limb . return of motor power was seen on postoperative day 6 and recovery of bladder functions on day 8 . a repeat mri scan done during second week showed significant reduction in size and number of intradural granulomas and resolution of arachnoiditis with normal cauda equina nerve roots . on postoperative day 15 , the patient was transferred to the ward with complete recovery of motor power of lower limbs and bladder functions , and with no residual sensory deficits . he was discharged from hospital a week later with advice to continue antitubercular treatment and to follow - up in neurology clinic . we describe a case of ces that occurred when a patient with previously undiagnosed tubercular arachnoiditis underwent an uneventful surgery for debridement of right foot under spinal anaesthesia with hyperbaric lignocaine . with the postoperative mri suggesting the diagnoses of tubercular arachnoiditis , later confirmed by a pcr test for tuberculosis , the patient was put on high - dose steroid therapy and antitubercular drugs , to which he responded very well , gaining complete neurological recovery in 2 weeks . in our review of the literature , we did not find any previous reports of diagnosed or undiagnosed tubercular arachnoiditis complicating spinal anaesthesia . although tubercular arachnoiditis is the most common type of infectious arachnoiditis in india , increasing incidence of the disease is being seen in the developed nations in conjunction with acquired immunodeficiency syndrome ( aids ) . frequent involvement of nerve root and spinal cord in the subarachnoid space differentiates tubercular arachnoiditis from arachnoiditis due to other causes like intrathecal administration of contrast agents , antibiotics , local anaesthetics ; traumatic punctures or bloody taps occurring after a neuraxial block ; trauma ; surgery ; disc disease ; etc . tubercular radiculomyelitis. tubercular arachnoiditis may occur as a primary lesion , or as secondary to tubercular meningitis or vertebral tuberculosis . most patients present with backache ( 86% ) and fever ( 67% ) , and smaller numbers with radicular pain , paraesthesias , subacute paraparesis , bladder and bowel disturbances , and vertebral kyphosis . in some cases in early stages of this disease , inflammation leads to the clumping of cauda equine nerve roots and granulomas formation . the later stages are marked by irregular csf loculations and blockage of csf flow due to formation of arachnoid bands . it has been seen that in patients receiving central neuraxial block , undetected underlying spinal pathologies such as spinal canal stenosis spinal metastatic lesion and subarachnoid cyst can cause ces due to the local anaesthetic drug being maldistributed in the compromised subarachnoid space . the maldistribution in turn causes high concentrations of the local anaesthetic drug in csf leading to structural and functional changes in the neural tissue . the same mechanism is responsible for ces from use of spinal microcatheter or with repeat spinal injections in the event of failed or patchy blocks . although ces has been reported with several local anaesthetics , it is frequently associated with lignocaine . yet lignocaine remains a useful drug for the procedure in short ambulatory surgery due to its fast onset , intense sensory and motor blocks , and rapid recovery , with dosage below 60 mg considered safe for subarachnoid block . other identified causes of ces after spinal anaesthesia include direct needle trauma , spinal cord ischaemia , abscess and spinal haematomas . cases of ces complicating spinal block have been typically seen in the immediate postoperative period , but these may occur later as well , as documented in two cases reports where ces due to subdural haematoma and epidural haematoma was seen 96 hours and 3 months respectively , after uneventful spinal anaesthesia . in the present case , we presume that the underlying disease pathology of tubercular arachnoiditis may have caused neurotoxicity of lignocaine by restricting the spread of the drug in the subarachnoid space , eventually resulting in ces , even though a safe dosage ( 50 mg lignocaine ) was used . besides , the use of small bore spinal needle , the hyperbaric drug , or the sitting position could have been additional contributory factors in the ces . however , direct damage to the spinal cord by dural puncture did not seem a likely factor as there was no neurological pain or dysethesia at the time of needle insertion . in conclusion , this case highlights that like any undetected pre - existing spinal pathology , asymptomatic tubercular arachnoiditis can contribute to ces even after carefully administered spinal anaesthesia . therefore , after surgery under spinal anaesthesia , careful monitoring for recovery of sensory and motor functions is important for early detection of a neurological complication such as ces . in a case where ces is suspected ( 1 ) the patient should be investigated thoroughly and an mri done to identify and suitably address any underlying contributory spinal pathology ; and ( 2 ) appropriate treatment should be immediately instituted for the same .

a number of programs have been developed for simulating population genetic and genetic epidemiological data conforming to one of three main algorithmic approaches : ' forwards ' , ' backwards ' and ' sideways ' . this review aims to make the reader aware of the range of options currently available to them . while no one program emerges as the best choice in all circumstances , we nominate a set of those which currently appear most promising .

the two main reasons for wanting to simulate genetic data are , first , to gain insight into the effects that underlying demographic and mutational parameters may have on the genetic data one sees , and , secondly , to create test datasets for assessing the power of alternative genetic analysis methods . ways of tackling the first goal range from informal approaches , which aim at getting a ' feel ' for how altering different parameters affects the output data , to more formal methods based on matching many simulated datasets to an observed dataset ( eg approximate bayesian computation ) . to tackle the second goal ( and particularly for genetic epidemiology methods ) , an additional ' ascertainment ' modelling element is often required to allow the simulation of disease - affecting loci within the context of a given study design ( such as a case - control study ) . the key challenges that all simulation algorithms face are : ( 1 ) speed -- typically one wants to do lots of simulations , so they need to be fast ; ( 2 ) scalability -- with the advent of genome - wide genotyping and large - scale sequencing , there is a need for simulation programs to match ; and ( 3 ) flexibility -- can the program cope with different demographic histories , population structure , recombination , selection , mutation models and disease models ? there are three main approaches to dealing with these challenges , here termed ' backwards ' , ' forwards ' and ' sideways ' . ' backwards ' ( or coalescent ) simulations start with the sample of individuals that will form your simulated dataset , then work backwards in time to construct the ancestral tree or graph of genealogical relationships that connects them all . the simulation algorithm does not actually have to work backwards in time to achieve this , but this is a technical detail . the important point is that by restricting attention just to the genealogical structure relevant to the sample in question , a large computational saving is generally achieved relative to the ' forwards - in - time ' approach . still greater efficiency is afforded by the classic coalescent approach , which employs a continuous - time approximation to effectively skip over the intermediate generations between important tree - generating events . ' forwards ' simulations start with the entire population of individuals -- typically , many thousands -- and then follow how all the genetic data in question are passed on from one generation to the next . one usually needs to simulate over many thousands of generations in order to arrive at an equilibrium in which the genetic characteristics of the population are independent of the original starting conditions . finally , ' sideways ' simulations start with a collection of real present - day genetic data , and use these as a template for generating new simulated data with similar properties . ' sideways ' algorithms can also be coalescent - based ( and thus fit into both ' backwards ' and ' sideways ' categories ) but some adopt simpler resampling strategies that do not explicitly consider changes over generational time in either direction . table 1 lists all programs that the authors were able to source via pubmed and other internet - based searches . ( coalescent ) approaches formthe largest part of table 1 , reflecting the inherent attractiveness and computational efficiency of simulating just that part of the genealogy needed to produce the data in the simulated sample . mshot , snpsim and cosi extend the algorithm to allow variable recombination rates along the dna sequence , and mshot , cosi , coasim and newgenecoal also allow ( allelic ) gene conversion in addition to crossovers as recombination events . simcoal introduces complex demographic models , simcoal2 extends this to variable recombination , serial simcoal allows sampling at multiple time points and modeler4simcoal provides a handy graphical user interface . flexible , but not necessarily easy to implement , coalescent simulators are provided by coasim , mlcoalsim , sarg and geneartisan . for neutral loci , the tree or graph - generating step can be conveniently decoupled from the mutation - generating step , and the latter can be run via a separate program such as andy rambaut 's seqgen program to produce a wide range of different types of genetic data from a range of different mutational models . it is also possible to decouple the sampling ascertainment process ( eg to get case - control data ) by applying this as an additional step to unascertained simulated data . currently , however , there are no easy ways of doing this , as additional user coding would be needed to adapt the sampling algorithms available in , for example , coasim , simupop[28 - 30 ] or fregene . furthermore , there are as yet no completely flexible ascertainment options that would allow , for example , simulation of cases from models with more than one partially linked disease locus , or from more general causal models that have incorporated additional covariates . this is because recombination gives rise to complex ancestral recombination graphs ( args ) rather than simple binary genealogical trees , and more recombination leads to ever larger and more complex args . the fastcoal and genome simulators employ approximations to the real coalescent - with - recombination that lead to simpler args and thence to feasible genome - wide simulations . macs , a recent update to fastcoal which uses an improved approximation to the coalescent - with - recombination , is available on request from jeff wall ( http://[email protected] ) . fastcoal is reported to be able to generate 2,000 50-megabase ( mb ) diploid samples in two minutes on a standard workstation , and genome to generate 600 150 mb diploid samples in 66 minutes . forwards - in - time simulators are more naturally capable of coping with complex modelling scenarios , at the expense of decreased computational efficiency . of these , the fregene and genomepop programs make the biggest effort at maintaining speed , and , of these , only fregene allows for ascertained disease - gene sampling . a useful scaling option in both programs allows one to simulate a smaller population over a smaller number of generations and then use these results to approximate a larger population over more generations . unfortunately , only diallelic snp data can be simulated fast enough to cover large genomic regions . at smaller genomic scales , more complex nucleotide and codon models can be simulated by genomepop , while copy number variation ( cnv ) and microsatellite data can be simulated by simupop [ 28 - 30 ] and nemo . genomesim claims to be able to generate genome - wide snp data by forwards simulation , but only achieves this by simulating over a very limited ten or so generations , far fewer than that needed to achieve proper genetic equilibrium . indeed , fregene and genomepop could also generate genome - wide datasets in this way , and presumably could do so with greater computational efficiency . sideways simulators can , to some extent , side - step the whole issue of model complexity by relying on real data ' as is ' to guide the simulation process . simple bootstrap resampling breaks down for longer regions because the genetic diversity seen in the reference sample ( usually the 270 individuals in hapmap ) is not adequate to capture the full diversity among all humans . the situation will improve with the ' 1,000 genomes ' project , and also with the steady increase in publically available genome - wide snp data , but it still seems sensible to apply an additional method to perturb the simulated data away from the narrow range seen in the real data . dudbridge proposed forming random diploid chromosomes from phased hapmap data followed by a single round of artificial meiosis , governed by empirical recombination rates also estimated from hapmap . this idea has been put to use in the hap - sample software , with an additional option to boost the baseline recombination rate ( x100 recommended ) to reduce long - range linkage disequilibrium . durrant et al . proposed an alternative idea based on sliding windows for introducing new variations into simulated data . this method has been implemented in the gwa simulator software , and an improved extension to this idea , which allows a variable sliding window size , has been implemented in the gs software . jonathan marchini 's hapgen software , based on the same underlying principles as his genotype imputation software impute , applies an approximation to the coalescent - with - recombination to generate new simulated data from existing phased hapmap data , but is slower than the other two sideways simulators . hap - sample is reported to be able to generate 2,000 samples of a 100,000 genome - wide snp chip in a few minutes on a standard workstation , and gs to generate 2,000 samples of chromosome 6 ( 36,000 snps ) in 140 minutes . in summary , no one program is capable of doing everything , but there exist some useful applications from all three main simulation approaches . for genome - wide snp data , the main contenders are fastcoal , genome , hap - sample and gs . for high model flexibility and sampling ascertainment at the 10 mb scale or less ( not whole - genome but still enough for many purposes ) , fregene is recommended . simulation of copy number variation and/or microsatellite data at larger genomic scales , and of more complex disease models allowing covariates and linked loci , remain areas for future program development .

background : traumatic brain injury ( tbi ) is a major public health problem . both genders are affected , but little is known about female tbi . the present study exclusively explores epidemiological , clinical , imaging , and death aspects of female tbi , and how it differs from males.methods:it is a retrospective study . data were documented from a tertiary institute during january 2010 to march 2010 . all variables were documented on standard proforma . the data were analyzed using r statistics software . age group was categorized into pediatric ( < 18 years ) , middle ( 1960 years ) and elderly ( > 61 years ) . significance was tested using chi - square test at the significance level of p < 0.05.results:data of 1627 tbi patients were recorded . of the total , female tbis contributed nearly 20% . compared to males , female patients reported higher percentages in manifesting symptoms ( 84.3% vs. 82.6% ) , injuries due to fall ( 32.1% vs. 24.4% ) , and surgical interventions ( 11.6% vs. 10.4% ) . female patients were significantly higher in mild head injury group ( 76.8% vs. 69.5% , p - 0.016 ) and mortality ( 3.4% vs. 1.6% , p - 0.048 ) . number of patients and deaths was more among females than males in pediatric and elderly age group . severities of injuries were more among female patients than male patients in middle and elder age groups.conclusion:the study results observe that female tbi group differ significantly in the severity of injury and mortality .

traumatic brain injury ( tbi ) is a major public health problem as evidenced by both clinicians and epidemiologist in india . disability due to neurological illness ranks third in india ; the major contributor is tbi . rapid increase in population , motorization , and industrialization in our country has contributed to the significant increase in tbi . tbi results in deaths , injuries , and disabilities of all age groups , especially young and productive people . gender has a significant effect on tbi , although both men and women experience brain injury there is the difference in them in the epidemiological , clinical , imaging , and outcome . majority of the studies on tbi emphasize more on male population as the incidence of tbi is more in male population . if stated , only few variables related to the female population are highlighted . in the literature , the characteristics or differences unique to female patients with tbi has not been explored in great detail . the present study scrutinizes epidemiological , clinical , imaging , treatment , and mortality variables exclusively among female tbi patients . the study also intends to identify any variable that differs between female and male population . this study was conducted in national institute of mental health and neurosciences , a tertiary - level referral center for neuroscience that has exclusive trauma care facility for tbi patients . the patients with brain injury are evaluated by neurosurgery residents and patients details are entered on a structured head injury pro forma . data of all patients with tbi who presented to casualty over a period ( from january 1 , 2010 , to march 15 , 2010 ) were retrospectively reviewed . the following variables were analyzed : demographic details such as age , gender , the cause of the injury ; clinical data like time since injury , injury severity , symptoms manifestation ; computed tomography ( ct ) scan findings such as intracranial bleed , skull fractures ; treatment like medical or surgical management ; and mortality at discharge . the data were categorized into pediatric ( < 18 years ) , middle age ( 1960 years ) , and elderly age ( > 61 years ) . the statistical analysis was performed in r statistics ( r.3.2.0 ) ( institute for statistics and mathematics of wu ( wirtschaftsuniversitt wien ) ) . data were expressed using descriptive statistics such as mean and standard deviation for continuous variables , and frequency and percentage for categorical variables . chi - square test was used among categorical data with p < 0.05 as significance . the statistical analysis was performed in r statistics ( r.3.2.0 ) ( institute for statistics and mathematics of wu ( wirtschaftsuniversitt wien ) ) . data were expressed using descriptive statistics such as mean and standard deviation for continuous variables , and frequency and percentage for categorical variables . chi - square test was used among categorical data with p < 0.05 as significance . during the study period , there were 1627 patients with head injuries . of them , 293/1627 ( 18% ) were female patients . the incidence of tbi was higher in first , fifth , and the sixth decade among the female patients as compared to males [ figure 1 ] . mean duration for the female patients to reach hospital was 18 h 27 min which was lesser than male patients ( 19 h 29 min ) . percentage of female and male brain injuries in decade wise the severity of brain injuries assessed by glasgow coma scale ( gcs ) among female patients revealed 225 ( 76.8% ) mild injuries , 37 ( 12.6% ) moderate and 31 ( 10.6% ) severe injuries . the manifesting symptoms among female tbis are ; loss of consciousness 152 ( 51.9% ) , vomiting in 154 ( 52.6% ) , ear or nose bleeding 71 ( 24.2% ) , seizures in 20 ( 6.8% ) , headache in 4 ( 1.4% ) , and limb weakness in 2 ( 0.7% ) . road traffic accidents was a major cause of injury 168 ( 57.3% ) , followed by falls 94 ( 32.1% ) , assaults 26 ( 8.9% ) , and others 5 ( 1.66% ) . among female patients head , the abnormal ct findings included parenchymal contusion 70 ( 23.9% ) , extradural hematoma 23 ( 7.8% ) , subdural hematoma subdural hemorrhage ( 14% ) , subarachnoid hemorrhage ( 4.4% ) , cerebral edema 72 ( 24.6% ) , petechial hemorrhage 5 ( 1.7% ) , ventricular hemorrhage 4 ( 1.4% ) , infarct 4 ( 1.4% ) , skull fracture 61 ( 20.8% ) , and pneumocephalus 10 ( 3.4% ) . majority of patients were managed medically , but however , 34 ( 11.6% ) patients required surgery for emergency hematoma evacuation . nearly , 33% of patients with severe tbi underwent surgery . tables 1a and b depict details of gcs , cause , symptoms , image findings , treatment , and outcome in both genders and different age groups . female and male pediatric , middle and elderly patients ; mode of injury , clinical symptoms , emergency evaluation and image findings with outcome female and male number and percentages of mode of injury , clinical symptoms , emergency evaluation , and image findings with outcome during the study period , there were 1627 patients with head injuries . of them , 293/1627 ( 18% ) were female patients . the incidence of tbi was higher in first , fifth , and the sixth decade among the female patients as compared to males [ figure 1 ] . mean duration for the female patients to reach hospital was 18 h 27 min which was lesser than male patients ( 19 h 29 min ) . percentage of female and male brain injuries in decade wise the severity of brain injuries assessed by glasgow coma scale ( gcs ) among female patients revealed 225 ( 76.8% ) mild injuries , 37 ( 12.6% ) moderate and 31 ( 10.6% ) severe injuries . the manifesting symptoms among female tbis are ; loss of consciousness 152 ( 51.9% ) , vomiting in 154 ( 52.6% ) , ear or nose bleeding 71 ( 24.2% ) , seizures in 20 ( 6.8% ) , headache in 4 ( 1.4% ) , and limb weakness in 2 ( 0.7% ) . road traffic accidents was a major cause of injury 168 ( 57.3% ) , followed by falls 94 ( 32.1% ) , assaults 26 ( 8.9% ) , and others 5 ( 1.66% ) . the abnormal ct findings included parenchymal contusion 70 ( 23.9% ) , extradural hematoma 23 ( 7.8% ) , subdural hematoma subdural hemorrhage ( 14% ) , subarachnoid hemorrhage ( 4.4% ) , cerebral edema 72 ( 24.6% ) , petechial hemorrhage 5 ( 1.7% ) , ventricular hemorrhage 4 ( 1.4% ) , infarct 4 ( 1.4% ) , skull fracture 61 ( 20.8% ) , and pneumocephalus 10 ( 3.4% ) . majority of patients were managed medically , but however , 34 ( 11.6% ) patients required surgery for emergency hematoma evacuation . nearly , 33% of patients with severe tbi underwent surgery . tables 1a and b depict details of gcs , cause , symptoms , image findings , treatment , and outcome in both genders and different age groups . female and male pediatric , middle and elderly patients ; mode of injury , clinical symptoms , emergency evaluation and image findings with outcome female and male number and percentages of mode of injury , clinical symptoms , emergency evaluation , and image findings with outcome our study summarizes that female population contributes to nearly one - fifth ( 18% ) of the total brain injuries . highest percentage of brain injuries are appreciated in the third decade ( 19.8% ) followed by fifth ( 17.5% ) and first decade ( 17% ) [ figure 1 ] . there was a significant difference in mortality between female ( 3.4% ) and male ( 1.6% ) . we found that there was larger proportion of females in age group of < 18 years . patients in middle age manifest with higher percent whereas pediatric and elderly age report the lesser percent of symptoms as compared with males . in pediatric and elderly age group , the road traffic injuries were lesser and falls were more in females as compared with males . among females only in elderly age group showed the higher percent of abnormal image findings . the mortality was more in all the age groups in females [ table 1a ] . the present study reports high male to female ratio that is 4.5:1 as males are common road users , and are predominantly affected in road traffic injuries and also most commonly involved in disputes . the high ratio of male to female injury is difficult to explain based on place of data acquisition as gender ratio is nearly same ( male : female , 1.1:1 ) . high male : female ( > 4:1 ) ratio was reported from a south african study . our study revealed a higher proportion of females in third decade followed by fifth and first decade . we found that nearly two - third of female patients were in a third to sixth decade . the reason might be that these age groups are more vulnerable to road injuries and disputes . studies from the usa , france , and eritrean have shown a higher incidence of brain injuries in female in first , second and third decade . age group < 18 years contributes to one - fourth percent of total injuries which is more than male pediatric group . high percent of mild head injuries are noted in pediatric tbi importantly in both genders , but in the current study proportion of females with tbi was higher ( 77% ) than male in mild injury category . significance in the severity of injury between female and male is documented with respect to specific risk factors . a meta - analysis observed that female patients with tbi report a higher number of trauma symptoms as compared to males . our study reports that female in third to sixth decade reports higher percentages of posttraumatic symptoms than males . the females in reproductive age group as an effect of hormonal influences have reported inconsistent posttraumatic symptoms . studies have reported that among pediatric and elderly age groups falls is the common mode of tbis . we could appreciate same in our results , but the percentage of female patients is more than male group . a study from eritrean has reported that an abnormal ct finding is about 54.5% in all the severities of brain injuries in both the gender . our study reports higher percent of abnormal ct findings ( 71.6% ) in all severities and both gender , but the elderly female category had higher percentage of abnormal ct findings than elderly male group . in our study , we found that female patients with severe tbi had higher requirement neurosurgical intervention than their male counterparts , which is similar to the findings from sweden study . the mortality report of the study demonstrates that the percentage of female group is significantly more than male group . tbi - induced death at 1 year follow - up study from scotland reports the percentage of deaths in female ( 13.5% , 21/156 ) is more than male ( 8.5% , 52/611 ) . proper plan for a prospective study with detailed variables will provide more insight . however , the present study provides basic details of tbi . a follow - up study documenting serial posttraumatic symptoms and quality of life would have been more effective . the present study comprises of 293 female tbi patients for a period of 2 months . an institute study from asmara , eritrea reports about 28 female injury patients for 5 months duration . a female domestic violence based study from three metropolitan cities report 169 patients for a period of 79 months . even though the sample size was not planned for the present study , the number of patients reported from a single institute for female tbi seems to be good for the mentioned duration as compared with existing literature . the present study highlights that even though the incidence of tbi is lesser in female population ; they have a higher percentage of abnormal image findings in elderly group , needs higher neurosurgical intervention and reports high number of deaths .

the purpose of this pictorial essay is to demonstrate the imaging features ( ultrasound , mammogram , and magnetic resonance imaging ( mri ) ) of alloderm(lifecell corp . ; branchburg , nj ) , an acellular dermal matrix sometimes used in both primary and reconstructive breast surgeries . alloderm is derived from cadaveric dermis and provides an immunologically inert scaffold in tissue reconstruction . since there is little literature on the imaging of this substance , radiologists may be unfamiliar with its appearance in breast imaging . for this manuscript , ex vivo and in vivo images of alloderm in postmastectomy patients were evaluated using different imaging modalities . the appearance of alloderm can vary based on length of time postsurgery and incorporation into the host . alloderm appears as an isodense to glandular tissue on a mammogram and isoechoic to glandular tissue on ultrasound imaging . on mri , in comparison with normal breast parenchyma , alloderm is hyperintense on t2-weighted imaging and isointense on t1-weighted imaging and demonstrates mild enhancement . to the best of the authors knowledge , this is the first multimodality imaging description of alloderm used in postmastectomy patients . the conformation of alloderm at surgical placement and the degree of host cell migration and neoangiogenesis are factors to take into consideration when performing diagnostic evaluations ; and , familiarity with the various imaging appearances of alloderm can be helpful to exclude residual or recurrent disease .

alloderm ( lifecell corp . ; branchburg , nj ) is an acellular dermal matrix originating from cadaveric dermis , which is processed to remove antigenic epitopes and cells , creating an immunologically inert scaffold . the matrix [ figure 1 ] contains collagen , elastin , hyaluronic acid , fibronectin , proteoglycans , growth factor receptors , and vascular channels , which allow for host cell migration and angiogenesis . in current practice , the pectoralis major muscle provides inadequate coverage for the tissue expander / implant , an alloderm sling described by breuing and warren is created by sewing it to the inferior edge of the pectoralis major muscle and along the chest wall at the location of the inframammary fold to construct a subpectoral - sub - allodermpocket . in the united states , over 75% of implant - based reconstruction is done using dermal matrices ( all name brands ) ; of that group over 75% is specifically alloderm . complications associated with alloderm including infection , hematoma formation , and flap necrosis are similar to those seen with standard submuscular reconstruction . a systematic review by sbitany and serletti , found a higher rate of postoperative seromas in patients undergoing reconstruction with acellular dermal matrix compared with those without . colwell et al . , found no significant difference in the total overall costs between single - stage implant reconstruction with alloderm and tissue - expander reconstruction without alloderm . breast imaging after reconstruction with alloderm is typically obtained for diagnostic evaluation when there is concern of a postoperative complication , or a clinical area of concern such as a palpable abnormality or pain . , reported a case of a patient with a new palpable , fixed mass in her breast after mastectomy . surgical excision of the mass demonstrated a foreign body giant cell infiltrate secondary to the acellular dermal matrix , without evidence of recurrent tumor . a literature search yielded a single preliminary report describing the radiologic appearances of alloderm in breast conservation therapy patients where a brief description of the imaging appearance of the postmastectomy alloderm sling was described by dialani as isointense to glandular tissue on t1w imaging and lacking enhancement with gadolinium . the objective of this manuscript is to provide imaging features of alloderm in both primary and reconstructive breast surgeries , particularly in the postmastectomy patient . ex vivo imaging of alloderm ( after hydrating in normal saline for 30 min as per usage instructions ) was performed with mammography [ figure 2 ] , ultrasound [ figure 3 ] , and magnetic resonance imaging ( mri ) [ figure 4 ] to illustrate the imaging features prior to host cell migration and angiogenesis . ( a ) a single sheet of alloderm ( asterisk ) was placed on the medial aspect of a breast phantom , and ( b ) a right craniocaudal view obtained shows the alloderm is isodense to . there are simulated calcifications ( arrows ) and two biopsy tracts ( arrowheads ) in the phantom . ( a ) transverse view shows a single sheet of alloderm overlying the skin in the field of view entirely . ( b ) transverse view of partially visualized alloderm adjacent to normal breast parenchyma , shows the alloderm is hyperechoic to normal breast parenchyma and demonstrated posterior acoustic shadowing . ( c ) additional transverse view of normal breast with and without alloderm on top of the breast ( junction denoted with an arrow ) was performed using a standoff gel pad reveals the hyperechoic surface of the alloderm . artifacts presumably caused by the microairbubbles within the acellular alloderm can be seen deep to the alloderm . noncontrast mri imaging with technique meeting acr - accreditation standards was performed on a ge signa hd 1.5 t magnet ( waukesha , wi ) . a folded sheet of alloderm was taped to the inferomedial aspect of the left breast ( arrows ) . fat saturated t1-weighted 3d spoiled gradient - recalled echo ( spgr ) ( a ) axial and ( b ) sagittal reformatted images show alloderm exhibiting hypointense - to - isointense t1 signal compared with glandular breast tissue . fat - suppressed t2-weighted ( c ) axial and ( d ) sagittal images of alloderm demonstrate hyperintense t2 signal with heterogeneity which is likely related to the folded configuration of the alloderm ; and ( e ) sagittal diffusion - weighted imaging ( dwi ) and ( f ) apparent diffusion coefficient ( adc ) images of alloderm show no restricted diffusion . intraoperative photos illustrate how alloderm can be used in reconstruction of the breast [ figure 5 ] . table 1 summarizes the imaging features of alloderm in our patients compared with those in the study by tran cao et al . intraoperative photos of alloderm ( asterisk ) placement and suturing initially ( a - d ) and ( e - h ) 4 -months later . ( a , b ) with the tissue expander in place , the alloderm is shown sewn to the pectoralis major muscle above and the inframammary fold below . ( c and d ) the alloderm is checked for contour and a hand in glove fit to the overlying skin envelope . ( e - h ) four months later , at a second stage , the patient undergoes implant exchange with removal of the tissue expander . just deep to the subcutaneous fat bleeding after incising the alloderm demonstrates host incorporation and vascularization ( e and f , arrow ) . the tissue expander ( g and h , arrow ) is exposed , and the undersurface of the incorporated alloderm can be visualized ( h , asterisk ) . mammogram : ( a ) right mediolateral oblique ( mlo ) and ( b ) right craniocaudal ( cc ) implant displaced views demonstrate multiple variable - sized circumscribed masses ( arrows ) that are isodense to normal glandular tissue and most prominent along the lateral aspect of the implant and the axillary tail . targeted ultrasound : ( c ) , without and ( d ) with color flow in the right axilla at the palpable area of concern , demonstrate multiple isoechoic , vague parallel hypoechoic to isoechoic masses with smooth margins ( c and d , arrows ) . ( e and f ) transverse and longitudinal sonographic views of the right breast demonstrate some of the masses resembling fatty lobules extending approximately 6 cm inferior to the lateral edge of the mastectomy scar to 7 cm superior to the lateral edge of the scar ( e and f , arrow ) . similar findings continue to extend toward the axilla adjacent to the implant ( images not shown ) . mri : fat - suppressed t2-weighted images ( g - i ) at three different axial slice locations ( superior to inferior ) show there is an elongated slightly lobulated area of increased t2 signal ( g - i , arrows ) in the right breast , ( j - l ) postgadolinium images at the same locations show no appreciable enhancement ( j - l , arrows ) corresponding to the location of the alloderm sling adjacent to an intact silicone implant , and , ( m - o ) post - gadolinium with cad overlay ( m - o ) show no kinetic enhancement associated with the alloderm . ( p ) diffusion - weighted image and ( q ) apparent diffusion coefficient map demonstrate mildly increased signal without evidence of restricted diffusion ( arrows ) . the mri findings related to the alloderm extended from the hammock / sling into the axilla where it was palpable . case 2 : 46-year - old female with bilateral prophylactic mastectomies and bilateral subpectoral implants . mri : contrast - enhanced ( a ) axial , ( b ) reformatted sagittal , and ( c ) postgadolinium with cad overlay images demonstrate mildly increased prominence and enhancement ( arrows ) posterior and lateral to an intact right implant . prior mri : contrast - enhanced a ) axial , b ) reformatted sagittal of the left breast , and c ) postgadolinium with cad overlay mr images demonstrate an area of minimal non - mass enhancement in the location of the alloderm ( arrows ) in the inframammary fold posterior and lateral to the implant . follow - up mri 16 months later : there are intact implants and less conspicuity and prominence of the non - mass enhancement ( d f , arrows ) comparison of imaging features of alloderm between study by tran cao et al . and this study a 36-year - old female , who 2 years prior underwent bilateral skin sparing mastectomies and implants for left breast invasive ductal carcinoma , presented with palpable nodularity along the upper outer aspects of her reconstructed right breast . a diagnostic mammogram and targeted ultrasound demonstrated multiple masses corresponding to the area of palpable concern . right mediolateral oblique ( mlo ) and right cranial - caudal ( cc ) implant displaced views demonstrated multiple variable - sized circumscribed masses that were isodense to normal glandular tissue and most prominent along the lateral aspect of the implant and the axillary tail [ figure 6a and b ] . ultrasound without and with color flow at the palpable area of concern in the right axilla demonstrated multiple , vague parallel hypoechoic to isoechoic masses with smooth margins [ figure 6c and d ] . in the right breast some of the masses resembled fatty lobules extending approximately 6 cm inferior to the lateral edge of the mastectomy scar to 7 cm superior to the lateral edge of the scar [ figure 6e and f ] . subsequent breast mri to further characterize the masses in the right breast and axilla was performed . fat - suppressed t2-weighted images at three different axial slice locations ( superior to inferior ) showed an elongated area of increased t2 signal in the reconstructed right breast [ figure 6g i ] . postgadolinium images at the same locations showed no appreciable enhancement corresponding to the location of the alloderm sling adjacent to an intact silicone implant [ figure 6j l ] . postgadolinium with computer aided detection ( cad ) overlay [ figure 6m o ] images also showed no areas of kinetic enhancement corresponding to the areas of alloderm . diffusion - weighted image [ figure 6p ] and apparent diffusion coefficient map [ figure 6q ] demonstrated mildly increased signal without evidence of restricted diffusion . the mri findings related to the alloderm extending from the hammock / sling into the axilla where it was palpable . review with the plastic surgeon confirmed that this location and configuration corresponded to the alloderm sling . therefore , this was given a birads 2 ( breast imaging reporting and data system ) benign finding and the patient is currently doing well without evidence of recurrent breast cancer . a 46-year - old female with bilateral prophylactic mastectomies and bilateral subpectoral implants presented to her plastic surgeon 9-months after surgery with pain in her left breast . breast mri was ordered for further diagnostic evaluation after targeted ultrasound revealed no correlative finding . contrast - enhanced axial [ figure 7a ] , reformatted sagittal [ figure 7b ] , and postgadolinium with cad overlay [ figure 7c ] mri demonstrated mildly increased prominence and enhancement posterior and lateral to an intact right implant . no abnormality was noted in the left breast to correspond to the patient 's area of pain . the pain and nodularity resolved and the patient is currently doing well with no complaints regarding her reconstruction . a 58-year - old female who underwent a right mastectomy for invasive ductal carcinoma 8 years prior and a left mastectomy for high grade ductal carcinoma in situ 2 years prior and subsequent bilateral implants presented with an area of cellulitis and clinically presumed fat necrosis in the right breast . breast mri done 16 months earlier demonstrated intact implants , mild enhancement along the left inframammary fold in the location of the alloderm , and no areas suspicious for malignancy in either breast . contrast - enhanced axial [ figure 8a ] , reformatted sagittal image of the left breast [ figure 8b ] , and postgadolinium with cad overlay [ figure 8c ] mri demonstrated an area of minimal non - mass enhancement in the location of the alloderm ( arrows ) in the inframammary fold posterior and lateral to the implant on the left . the patient continued to have a mild tenderness over the medial aspect of her left breast . follow - up mri 16 months later the images showed intact implants and less conspicuity and prominence of the non - mass enhancement [ figure 8d f , arrows ] ex vivo imaging of alloderm ( after hydrating in normal saline for 30 min as per usage instructions ) was performed with mammography [ figure 2 ] , ultrasound [ figure 3 ] , and magnetic resonance imaging ( mri ) [ figure 4 ] to illustrate the imaging features prior to host cell migration and angiogenesis . ( a ) a single sheet of alloderm ( asterisk ) was placed on the medial aspect of a breast phantom , and ( b ) a right craniocaudal view obtained shows the alloderm is isodense to . there are simulated calcifications ( arrows ) and two biopsy tracts ( arrowheads ) in the phantom . ( a ) transverse view shows a single sheet of alloderm overlying the skin in the field of view entirely . ( b ) transverse view of partially visualized alloderm adjacent to normal breast parenchyma , shows the alloderm is hyperechoic to normal breast parenchyma and demonstrated posterior acoustic shadowing . ( c ) additional transverse view of normal breast with and without alloderm on top of the breast ( junction denoted with an arrow ) was performed using a standoff gel pad reveals the hyperechoic surface of the alloderm . artifacts presumably caused by the microairbubbles within the acellular alloderm can be seen deep to the alloderm . noncontrast mri imaging with technique meeting acr - accreditation standards was performed on a ge signa hd 1.5 t magnet ( waukesha , wi ) . a folded sheet of alloderm was taped to the inferomedial aspect of the left breast ( arrows ) . fat saturated t1-weighted 3d spoiled gradient - recalled echo ( spgr ) ( a ) axial and ( b ) sagittal reformatted images show alloderm exhibiting hypointense - to - isointense t1 signal compared with glandular breast tissue . fat - suppressed t2-weighted ( c ) axial and ( d ) sagittal images of alloderm demonstrate hyperintense t2 signal with heterogeneity which is likely related to the folded configuration of the alloderm ; and ( e ) sagittal diffusion - weighted imaging ( dwi ) and ( f ) apparent diffusion coefficient ( adc ) images of alloderm show no restricted diffusion . intraoperative photos illustrate how alloderm can be used in reconstruction of the breast [ figure 5 ] . table 1 summarizes the imaging features of alloderm in our patients compared with those in the study by tran cao et al . intraoperative photos of alloderm ( asterisk ) placement and suturing initially ( a - d ) and ( e - h ) 4 -months later . ( a , b ) with the tissue expander in place , the alloderm is shown sewn to the pectoralis major muscle above and the inframammary fold below . ( c and d ) the alloderm is checked for contour and a hand in glove fit to the overlying skin envelope . ( e - h ) four months later , at a second stage , the patient undergoes implant exchange with removal of the tissue expander . just deep to the subcutaneous fat the incorporated alloderm is seen ( e , asterisk ) . bleeding after incising the alloderm demonstrates host incorporation and vascularization ( e and f , arrow ) . the tissue expander ( g and h , arrow ) is exposed , and the undersurface of the incorporated alloderm can be visualized ( h , asterisk ) . mammogram : ( a ) right mediolateral oblique ( mlo ) and ( b ) right craniocaudal ( cc ) implant displaced views demonstrate multiple variable - sized circumscribed masses ( arrows ) that are isodense to normal glandular tissue and most prominent along the lateral aspect of the implant and the axillary tail . targeted ultrasound : ( c ) , without and ( d ) with color flow in the right axilla at the palpable area of concern , demonstrate multiple isoechoic , vague parallel hypoechoic to isoechoic masses with smooth margins ( c and d , arrows ) . ( e and f ) transverse and longitudinal sonographic views of the right breast demonstrate some of the masses resembling fatty lobules extending approximately 6 cm inferior to the lateral edge of the mastectomy scar to 7 cm superior to the lateral edge of the scar ( e and f , arrow ) . similar findings continue to extend toward the axilla adjacent to the implant ( images not shown ) . mri : fat - suppressed t2-weighted images ( g - i ) at three different axial slice locations ( superior to inferior ) show there is an elongated slightly lobulated area of increased t2 signal ( g - i , arrows ) in the right breast , ( j - l ) postgadolinium images at the same locations show no appreciable enhancement ( j - l , arrows ) corresponding to the location of the alloderm sling adjacent to an intact silicone implant , and , ( m - o ) post - gadolinium with cad overlay ( m - o ) show no kinetic enhancement associated with the alloderm . ( p ) diffusion - weighted image and ( q ) apparent diffusion coefficient map demonstrate mildly increased signal without evidence of restricted diffusion ( arrows ) . the mri findings related to the alloderm extended from the hammock / sling into the axilla where it was palpable . case 2 : 46-year - old female with bilateral prophylactic mastectomies and bilateral subpectoral implants . mri : contrast - enhanced ( a ) axial , ( b ) reformatted sagittal , and ( c ) postgadolinium with cad overlay images demonstrate mildly increased prominence and enhancement ( arrows ) posterior and lateral to an intact right implant . prior mri : contrast - enhanced a ) axial , b ) reformatted sagittal of the left breast , and c ) postgadolinium with cad overlay mr images demonstrate an area of minimal non - mass enhancement in the location of the alloderm ( arrows ) in the inframammary fold posterior and lateral to the implant . follow - up mri 16 months later : there are intact implants and less conspicuity and prominence of the non - mass enhancement ( d f , arrows ) comparison of imaging features of alloderm between study by tran cao et al . and this study a 36-year - old female , who 2 years prior underwent bilateral skin sparing mastectomies and implants for left breast invasive ductal carcinoma , presented with palpable nodularity along the upper outer aspects of her reconstructed right breast . a diagnostic mammogram and right mediolateral oblique ( mlo ) and right cranial - caudal ( cc ) implant displaced views demonstrated multiple variable - sized circumscribed masses that were isodense to normal glandular tissue and most prominent along the lateral aspect of the implant and the axillary tail [ figure 6a and b ] . ultrasound without and with color flow at the palpable area of concern in the right axilla demonstrated multiple , vague parallel hypoechoic to isoechoic masses with smooth margins [ figure 6c and d ] . in the right breast some of the masses resembled fatty lobules extending approximately 6 cm inferior to the lateral edge of the mastectomy scar to 7 cm superior to the lateral edge of the scar [ figure 6e and f ] . subsequent breast mri to further characterize the masses in the right breast and axilla was performed . fat - suppressed t2-weighted images at three different axial slice locations ( superior to inferior ) showed an elongated area of increased t2 signal in the reconstructed right breast [ figure 6g i ] . postgadolinium images at the same locations showed no appreciable enhancement corresponding to the location of the alloderm sling adjacent to an intact silicone implant [ figure 6j l ] . postgadolinium with computer aided detection ( cad ) overlay [ figure 6m o ] images also showed no areas of kinetic enhancement corresponding to the areas of alloderm . diffusion - weighted image [ figure 6p ] and apparent diffusion coefficient map [ figure 6q ] demonstrated mildly increased signal without evidence of restricted diffusion . the mri findings related to the alloderm extending from the hammock / sling into the axilla where it was palpable . review with the plastic surgeon confirmed that this location and configuration corresponded to the alloderm sling . therefore , this was given a birads 2 ( breast imaging reporting and data system ) benign finding and the patient is currently doing well without evidence of recurrent breast cancer . a 46-year - old female with bilateral prophylactic mastectomies and bilateral subpectoral implants presented to her plastic surgeon 9-months after surgery with pain in her left breast . breast mri was ordered for further diagnostic evaluation after targeted ultrasound revealed no correlative finding . contrast - enhanced axial [ figure 7a ] , reformatted sagittal [ figure 7b ] , and postgadolinium with cad overlay [ figure 7c ] mri demonstrated mildly increased prominence and enhancement posterior and lateral to an intact right implant . no abnormality was noted in the left breast to correspond to the patient 's area of pain . the pain and nodularity resolved and the patient is currently doing well with no complaints regarding her reconstruction . a 58-year - old female who underwent a right mastectomy for invasive ductal carcinoma 8 years prior and a left mastectomy for high grade ductal carcinoma in situ 2 years prior and subsequent bilateral implants presented with an area of cellulitis and clinically presumed fat necrosis in the right breast . breast mri done 16 months earlier demonstrated intact implants , mild enhancement along the left inframammary fold in the location of the alloderm , and no areas suspicious for malignancy in either breast . contrast - enhanced axial [ figure 8a ] , reformatted sagittal image of the left breast [ figure 8b ] , and postgadolinium with cad overlay [ figure 8c ] mri demonstrated an area of minimal non - mass enhancement in the location of the alloderm ( arrows ) in the inframammary fold posterior and lateral to the implant on the left . , the patient continued to have a mild tenderness over the medial aspect of her left breast . follow - up mri 16 months later the images showed intact implants and less conspicuity and prominence of the non - mass enhancement [ figure 8d f , arrows ] . a 36-year - old female , who 2 years prior underwent bilateral skin sparing mastectomies and implants for left breast invasive ductal carcinoma , presented with palpable nodularity along the upper outer aspects of her reconstructed right breast . a diagnostic mammogram and right mediolateral oblique ( mlo ) and right cranial - caudal ( cc ) implant displaced views demonstrated multiple variable - sized circumscribed masses that were isodense to normal glandular tissue and most prominent along the lateral aspect of the implant and the axillary tail [ figure 6a and b ] . ultrasound without and with color flow at the palpable area of concern in the right axilla demonstrated multiple , vague parallel hypoechoic to isoechoic masses with smooth margins [ figure 6c and d ] . in the right breast some of the masses resembled fatty lobules extending approximately 6 cm inferior to the lateral edge of the mastectomy scar to 7 cm superior to the lateral edge of the scar [ figure 6e and f ] . subsequent breast mri to further characterize the masses in the right breast and axilla was performed . fat - suppressed t2-weighted images at three different axial slice locations ( superior to inferior ) showed an elongated area of increased t2 signal in the reconstructed right breast [ figure 6g i ] . postgadolinium images at the same locations showed no appreciable enhancement corresponding to the location of the alloderm sling adjacent to an intact silicone implant [ figure 6j l ] . postgadolinium with computer aided detection ( cad ) overlay [ figure 6m o ] images also showed no areas of kinetic enhancement corresponding to the areas of alloderm . diffusion - weighted image [ figure 6p ] and apparent diffusion coefficient map [ figure 6q ] demonstrated mildly increased signal without evidence of restricted diffusion . the mri findings related to the alloderm extending from the hammock / sling into the axilla where it was palpable . review with the plastic surgeon confirmed that this location and configuration corresponded to the alloderm sling . therefore , this was given a birads 2 ( breast imaging reporting and data system ) benign finding and the patient is currently doing well without evidence of recurrent breast cancer . a 46-year - old female with bilateral prophylactic mastectomies and bilateral subpectoral implants presented to her plastic surgeon 9-months after surgery with pain in her left breast . breast mri was ordered for further diagnostic evaluation after targeted ultrasound revealed no correlative finding . contrast - enhanced axial [ figure 7a ] , reformatted sagittal [ figure 7b ] , and postgadolinium with cad overlay [ figure 7c ] mri demonstrated mildly increased prominence and enhancement posterior and lateral to an intact right implant . no abnormality was noted in the left breast to correspond to the patient 's area of pain . the pain and nodularity resolved and the patient is currently doing well with no complaints regarding her reconstruction . a 58-year - old female who underwent a right mastectomy for invasive ductal carcinoma 8 years prior and a left mastectomy for high grade ductal carcinoma in situ 2 years prior and subsequent bilateral implants presented with an area of cellulitis and clinically presumed fat necrosis in the right breast . breast mri done 16 months earlier demonstrated intact implants , mild enhancement along the left inframammary fold in the location of the alloderm , and no areas suspicious for malignancy in either breast . contrast - enhanced axial [ figure 8a ] , reformatted sagittal image of the left breast [ figure 8b ] , and postgadolinium with cad overlay [ figure 8c ] mri demonstrated an area of minimal non - mass enhancement in the location of the alloderm ( arrows ) in the inframammary fold posterior and lateral to the implant on the left . , the patient continued to have a mild tenderness over the medial aspect of her left breast . follow - up mri 16 months later the images showed intact implants and less conspicuity and prominence of the non - mass enhancement [ figure 8d f , arrows ] given the differences in alloderm usage in this study and the one by tran cao et al . the earlier manuscript described alloderm folded into a roll to fill large lumpectomy defects ; this manuscript describes alloderm used as a hammock / sling in postmastectomy patients . the variability of sonographic features of alloderm between the two manuscripts and between the ex vivo and in vivo cases here probably reflects the continuum of vascularization and incorporation of alloderm into the host . in particular , the distinct contrast between the in vivo and ex vivo sonographic features suggests more posterior acoustic shadowing when there is less incorporation into the host . similarly , varying enhancing mri features shown in our patients compared with those of tran cao et al . can be attributed to differences in alloderm use and the elapsed time of incorporation into the host . we believe that mri enhancement of alloder varies over time , with little if any enhancement of the matrix initially , as demonstrated by tran cao et al . differentiating alloderm from abscess , recurrence , postoperative seroma / hematoma , fat necrosis , capsular contraction , or even extracapsular implant rupture is not always straightforward . in these settings , the imaging features of alloderm are nonspecific with alloderm being considered in the differential when it can be corroborated with the operative notes . in our practice , postmastectomy and postreconstructed breast concerns are addressed on a case - by - case basis . almost always , targeted ultrasound is the initial imaging modality of choice . for cases where fat necrosis is a differential consideration , mammograms may also be obtained to look for expected calcifications or for fat density masses . for breast reconstructions with a transverse rectus abdominus myocutaneous flap or deep inferior epigastric perforator flap , an initial mammographic approach is used when patients present with a clinical area of concern . ultrasound and mammograms are certainly the most cost - effective way for evaluation in these instances . many of the bilateral postmastectomy patients are initially referred to mri because of lack of breast parenchyma , the exquisite soft tissue characterization afforded by mri , and ability to simultaneously evaluate implant integrity . in those cases where conventional diagnostic workup with mammogram and ultrasound remains equivocal , mri is often performed , mainly because the conformation and configuration of alloderm relative to the rest of the reconstructed breast can be better assessed . through all our cases , we have found that the most helpful information comes from clinical history , correlation with the operative note , and direct discussion with the clinician or surgeon . in most cases , if there is no strong clinical suspicion and the patient is able to return for short - term follow - up , biopsy can thus be obviated . we suspect there is a continuum of imaging features of alloderm as it is incorporated into the host . a prospective longitudinal study at various time points of alloderm integration would be helpful . the variation in appearances can be confounded by the extent of incorporation of alloderm into the host at the time of imaging . familiarity of some of the appearances of alloderm in the postmastectomy reconstructed breast may help to obviate the need for immediate biopsy .

background : it has been stated that the bone allografts from different tissue banks may lead to various amount of bone induction , so the aim of this study was to evaluate bone regeneration of three demineralized allografts both histologically and histomorphometrically in rabbits calvaria bone defects.materials and methods : in this double - blind randomized experimental animal study , 32 critical size defects ( 11-mm diameter ) in the calvaria of 16 male new zealand white rabbits were randomly filled with three demineralized freeze - dried bone allografts ( dbm , cenobone , dembone ) , while the nongrafted defect was regarded as control group . after 6 and 12 weeks of healing , the experimental animals were euthanized for specimen preparation . after histological evaluation , histomorphometric analysis was performed to quantify new bone formation and remained graft particles . the data were analyzed by one - way anova with tukey 's ad - hoc test and t - test . ( p < 0.05 was considered to be statistically significant).results : mean percentage of bone formation increased between two healing time , but it was not statistically significant in all groups except dbm which the bone formation significantly decreased ( p = 0.04 ) . there were not statistically significant differences between three allografts in remained particles and bone formation in both healing times and they could not induce significantly more bone formation than control group.conclusion:both test and control groups resulted in successful new bone formation . no difference was noted in bone formation and remained particles between three commercial bone allografts . further studies in this issue may be needed .

lack of adequate bone is a common complication in periodontally compromised teeth and implant dentistry . autogenous bone is still the gold standard in bone augmentation procedures but its low availability and donor site morbidity necessitates the development of alternative products for it . many bone substitutes are introduced every day such as allografts , xenografts and synthetically produced ones . one of the commonly used substitute is allogenic bone graft . the use of demineralized freeze - dried bone allograft ( dfdba ) , whether alone or in combination with other bone substitute , showed significant improvements in bone augmentation procedures . in fact the presence of bone morphogenic proteins ( bmp ) in dfdba facilitates new bone formation by allowing undifferentiated mesenchymal progenitor cells undergo phenotypic conversion to the osteoblasts . the main advantage of allografts is that they eliminate the need for a donor site besides it can be used in large quantities if necessary . but there is a controversy about the effectiveness of bone allografts in bone regeneration between studies . becker et al . did not find that dfdba was beneficial for periodontal regeneration , while in the other study the use of dfdba improve the repair of periodontal lesions . there are different batches of allografts commercially available , but they might be different in bone inductive activity ( bmp concentration ) , depends on biological properties of the graft , criteria for selecting donors and methods of allograft processing . schwartz and colleagues stated that so many differences exist in bone bank preparations of dfdba and they can induce bone formation variously . however , the dental practitioners need to select the most efficient and cost effective ones for the routine dental practice . in fact the most effective products are those that maintain the porous structure and anatomy of mineralized bone with scrutinized sterilization that adhere to american association of tissue banks ( aatb ) guidelines . the aim of this study was to evaluate the ability of three different commercial dfdba to induce new bone formation in rabbit 's calvaria . sixteen new zealand white male rabbits weighing 2.0 - 3.0 kg were selected for this study ( they were mature skeletally ) . the rabbits were allowed to acclimatize 14 days before the experimental study . the animals were housed in separate cages under standard laboratory conditions and fed with a standard diet . animal selection , management , surgical protocol , and preparation were approved by the institutional animal care and use committee , torabinejad dental research center , school of dentistry , isfahan university of medical sciences , isfahan , iran . the animals were anesthetized by intramascular injection of 50 mg / kg of ketamine hydrochloride ( ketamine , alfasan , woerden , holland ) and 1 mg / kg of acepromazine ( neurotranq , alfasan , woerden , holland ) . the surgical sites shaved and then disinfected with alcohol and povidone iodine , followed by local anesthesia with 2% lidocaine hcl with epinephrine ( dilution 1:100,000 ) , ( persocaine - e , darou pakhsh pharmaceutical mfg.co , tehran , iran ) . an incision was made along the midsagittal suture from the frontal bone to the occipital bone . one standardized circular and bicortical defect with 11 mm diameter was created using trephine bur under constant cool - saline irrigation on each side of midsagittal suture [ figure 1 ] . thirty - two critical size defects were randomly filled with three dfdba : photograph of two standardized circular defects created with diameter of 11 mm dfdba 1(dbm , iranian tissue bank research and preparation center , tehran , iran ) particle size : 420 - 840 m , dfdba 2 ( cenobone , tissue regeneration corporation , kish , iran ) particle size : 500 - 1000 m and dfdba 3(dembone , pacific coast tissue bank , los angeles , usa ) particle size : 250 - 850 m . in control group , periosteum was sutured with a resorbable suture material ( 4 - 0 polyglycolate , hur - teb medical devices , ghazvin , iran ) and skin with ( silk 3 - 0 supasil , supa medical devices , tehran , iran ) . postoperative cares included the intramascular administration of antibiotic ceftriaxone 5 mg / kg ( ceftrax , jaberebne haian pharmaceutical mfg co , tehran , iran ) and the careful clinical observation of the animals throughout the healing period . the animals were sacrificed 6 and 12 weeks postoperatively using intracardial injection of magnesium sulfate under deep anesthesia . the area of the surgical defects and surrounding tissues were removed en bloc after sacrifice . the sections were decalcified in 10% formic acid solution for 20 days then dehydrated with grated alcohols and embedded in paraffin . the most - central sections ( the greatest diameter of the circle ) from each block were stained with hematoxylin and eosin ( h & e ) and examined using light microscopy ( nicon , e400 , japan ) [ figure 2 ] . ( a ) dbm at 6 weeks , ( b ) dbm at 12 weeks , ( c ) cenobone at 6 weeks , ( d ) cenobone at 12 weeks , ( e ) dembone at 6 weeks , ( f ) dembone at 12 weeks , ( g ) control at 6 weeks , ( h ) control at 12 weeks the central sections were chosen for histomorphometric analysis . computer - assisted histomorphometric measurements of the newly formed bone were obtained using an automated image analysis software . the new bone formation values , which were the percentile ratio of newly formed bone area over the total defect area , remained particles , percentage and type of inflammation , type of bone and connective tissue , were assessed by a blinded pathologist . significant differences among groups were identified by one - way anova with tukey 's ad - hoc test and significant differences among two healing times were determined by t - test ( p < 0.05 was considered to be statistically significant ) . the animals were anesthetized by intramascular injection of 50 mg / kg of ketamine hydrochloride ( ketamine , alfasan , woerden , holland ) and 1 mg / kg of acepromazine ( neurotranq , alfasan , woerden , holland ) . the surgical sites shaved and then disinfected with alcohol and povidone iodine , followed by local anesthesia with 2% lidocaine hcl with epinephrine ( dilution 1:100,000 ) , ( persocaine - e , darou pakhsh pharmaceutical mfg.co , tehran , iran ) . an incision was made along the midsagittal suture from the frontal bone to the occipital bone . one standardized circular and bicortical defect with 11 mm diameter was created using trephine bur under constant cool - saline irrigation on each side of midsagittal suture [ figure 1 ] . thirty - two critical size defects were randomly filled with three dfdba : photograph of two standardized circular defects created with diameter of 11 mm dfdba 1(dbm , iranian tissue bank research and preparation center , tehran , iran ) particle size : 420 - 840 m , dfdba 2 ( cenobone , tissue regeneration corporation , kish , iran ) particle size : 500 - 1000 m and dfdba 3(dembone , pacific coast tissue bank , los angeles , usa ) particle size : 250 - 850 m . in control group , the defects were filled with no bone material . periosteum was sutured with a resorbable suture material ( 4 - 0 polyglycolate , hur - teb medical devices , ghazvin , iran ) and skin with ( silk 3 - 0 supasil , supa medical devices , tehran , iran ) . postoperative cares included the intramascular administration of antibiotic ceftriaxone 5 mg / kg ( ceftrax , jaberebne haian pharmaceutical mfg co , tehran , iran ) and the careful clinical observation of the animals throughout the healing period . the animals were sacrificed 6 and 12 weeks postoperatively using intracardial injection of magnesium sulfate under deep anesthesia . the area of the surgical defects and surrounding tissues were removed en bloc after sacrifice . the sections were decalcified in 10% formic acid solution for 20 days then dehydrated with grated alcohols and embedded in paraffin . the most - central sections ( the greatest diameter of the circle ) from each block were stained with hematoxylin and eosin ( h & e ) and examined using light microscopy ( nicon , e400 , japan ) [ figure 2 ] . ( a ) dbm at 6 weeks , ( b ) dbm at 12 weeks , ( c ) cenobone at 6 weeks , ( d ) cenobone at 12 weeks , ( e ) dembone at 6 weeks , ( f ) dembone at 12 weeks , ( g ) control at 6 weeks , ( h ) control at 12 weeks computer - assisted histomorphometric measurements of the newly formed bone were obtained using an automated image analysis software . the new bone formation values , which were the percentile ratio of newly formed bone area over the total defect area , remained particles , percentage and type of inflammation , type of bone and connective tissue , were assessed by a blinded pathologist . significant differences among groups were identified by one - way anova with tukey 's ad - hoc test and significant differences among two healing times were determined by t - test ( p < 0.05 was considered to be statistically significant ) . mean regenerated bone and remained particles in the study samples are mentioned in table 1 . mean ( sds ) percentage of regenerated bone and remaining material in each study sample in all of the study groups the regenerated bone consisted of woven and lamellar bone was produced after 6 and 12 weeks . bone formation was seen from margin of the defects with a fibrous connective tissue at the center of the defects in dbm and control groups . there was bone formation in the margin and center of the defect in a fibrous connective tissue background in both healing times in cenobone , but only after 12 weeks in dembone allograft . there were foreign body reaction and chronic inflammatory cells in dbm samples in both healing times , but it was diminished in cenobone and dembone groups from 6 to 12 weeks and inflammatory aggregations were seen around the remained particles . chronic inflammatory cell aggregations could be seen around the remained particles in all tested allografts . mean regenerated bone was increased in dembone ( p = 0.40 ) , cenobone ( p = 0.12 ) and control ( p = 0.05 ) groups but significantly was decreased in dbm ( p = 0.04 ) from 6 to 12 weeks . there was a reduction in mean remained particles after 12 weeks in all allografts , but it was not statistically significant [ dbm ( p = 0.53 ) , cenobone ( p = 0.22 ) , dembone ( p = 0.009 ) ] . chronic inflammatory cells significantly was decreased during study period in all groups [ cenobone ( p = 0.01 ) , dembone ( p = 0.04 ) , control ( p = 0.01 ) ] except dbm which had the most inflammatory cells after 12 weeks between allografts . spearman rank correlation coefficient showed that there was a significant reverse relation between bone formation and remained particles in all groups ( p < 0.001 , r = 0.624 ) . comparison of mean percentage of bone formation and remained particles between study groups at both healing times are shown in figures 3 and 4 . comparison of bone formation in study groups at two healing times comparison of remained particles in study groups at two healing times after 6 weeks , cenobone has the least bone formation , even significantly less than control group ( p = 0.04 ) and there were not statistically significant differences between three allografts in remained particles and bone formation . after 12 weeks , all the bone grafts had more bone formation than 6 weeks except dbm , which demonstrated significantly less bone formation than control group ( p = 0.02 ) , besides it had the most remained particles between allografts after 3 months . there were not statistically significant differences between three allografts in remained particles and bone formation in this healing time too . in all of the study groups the regenerated bone consisted of woven and lamellar bone was produced after 6 and 12 weeks . bone formation was seen from margin of the defects with a fibrous connective tissue at the center of the defects in dbm and control groups . there was bone formation in the margin and center of the defect in a fibrous connective tissue background in both healing times in cenobone , but only after 12 weeks in dembone allograft . there were foreign body reaction and chronic inflammatory cells in dbm samples in both healing times , but it was diminished in cenobone and dembone groups from 6 to 12 weeks and inflammatory aggregations were seen around the remained particles . chronic inflammatory cell aggregations could be seen around the remained particles in all tested allografts . mean regenerated bone was increased in dembone ( p = 0.40 ) , cenobone ( p = 0.12 ) and control ( p = 0.05 ) groups but significantly was decreased in dbm ( p = 0.04 ) from 6 to 12 weeks . there was a reduction in mean remained particles after 12 weeks in all allografts , but it was not statistically significant [ dbm ( p = 0.53 ) , cenobone ( p = 0.22 ) , dembone ( p = 0.009 ) ] . chronic inflammatory cells significantly was decreased during study period in all groups [ cenobone ( p = 0.01 ) , dembone ( p = 0.04 ) , control ( p = 0.01 ) ] except dbm which had the most inflammatory cells after 12 weeks between allografts . spearman rank correlation coefficient showed that there was a significant reverse relation between bone formation and remained particles in all groups ( p < 0.001 , r = 0.624 ) . comparison of mean percentage of bone formation and remained particles between study groups at both healing times are shown in figures 3 and 4 . comparison of bone formation in study groups at two healing times comparison of remained particles in study groups at two healing times after 6 weeks , cenobone has the least bone formation , even significantly less than control group ( p = 0.04 ) and there were not statistically significant differences between three allografts in remained particles and bone formation . after 12 weeks , all the bone grafts had more bone formation than 6 weeks except dbm , which demonstrated significantly less bone formation than control group ( p = 0.02 ) , besides it had the most remained particles between allografts after 3 months . there were not statistically significant differences between three allografts in remained particles and bone formation in this healing time too . using enough information about the capacity of bone regeneration of these materials could help to select the most efficient and cost - effective ones . in this double - blind randomized experimental animal study , the amounts of regenerated bone and remaining graft material , along with the severity of inflammation and foreign body reactions , compared between groups of critical size defects grafted with three common allografts and it is recommended that a healing period of 8 weeks or more and the critical size defects of 11 mm or more should be used for the evaluation of late healing , such as resorption of materials and the amount of bone regeneration , in rabbit 's calvaria . there were not statistically significant differences between 3 allografts in remained particles and bone formation at two healing times and they could not induce bone formation significantly more than control group . found that dfdba showed higher bone formation than control group after 4 , 8 and 12 weeks but the defects were created with trephine 8 in the rat 's calvaria , which was different from this study . becker and colleagues did not find that dfdba was beneficial for periodontal regeneration and bone regeneration around implants , while abolfazli and colleagues found the use of dfdba improve the repair of periodontal lesions in two or three walls alveolar bone defects and reported that it had similar effect like autogenous bone on bone formation . in some studies schwartz and colleagues stated that so many differences exist in bone bank preparations of dfdba and they can induce bone formation variously . in this study dfdba from two banks caused new bone formation just after 2 months and dfdba from one of the tissue bank did not induce bone formation at all . in our study the defect closure and the new bone area ratio gradually increased with the healing time , but these parameters did not differ significantly between 6 and 12 weeks in all groups except dbm which the mean percentage of bone formation significantly decreased ( p = 0.04 ) between 2 healing times . this may be due to the chronic inflammation presented around the remained particles which was remained more than other allografts after 3 months . as we know the presence of inflammation is mandatory for bone healing but persistence of inflammatory mediators may lead to suboptimal bone formation . stated that the inflammation was reduced during healing time which is in agreement with the result of this study . dembone had the least inflammation between allografts after 6 weeks and there were not statistically significant differences between it and control group ( control group had minute inflammation at both healing times ) . although hydrochloric acid is needed for removal the masking effect of mineralized matrix on bmps , but putting the allografts for a long time ( more than 90 min ) in acid bath can affect the bmp concentration reversely . in fact the studies had shown that 2% residual calcium level is necessary for bone induction of allografts , in other words this level of calcium is optimal for osteoclastic resorption and following osteoblastic activity . besides herold et al . stated that we can find the highest alkaline phosphatase activity in cultured human periosteal cells with 2% residual calcium which is optimal for bone regeneration . the time of acid demineralization may be variant in different tissue banks and this may affect the osteoinductive and osteoconductive properties of them . origination of the healing from defect margin is constant and independent of using bone graft as we could see in control group . although cenobone had the least bone formation at 6 weeks , but there were bone islets at the centre of the defects which could be seen in defects grafted with dembone after 12 weeks . presence of bony islets at the center of the defect might present that the particles far from the margin were lined by osteoblasts and actively secret osteoid and this probably point to a more osteoconductive property of these allografts . in dbm specimens , we could see marginal bone formation , which means that the graft particles only near the host bone were involved in bone regeneration . speed of bone regeneration is important in some treatment modalities such as immediate loading . in this study we could not see any differences in bone formation between allografts at 6 weeks , so no allograft could be faster in bone formation than the other . the remained particles decreased with increasing bone formation and dembone had the least remained particles after 12 weeks which can be attributed to the nearly smaller size of the graft particles and this could justify the least inflammation of these samples between allografts at 12 weeks . according to the study done by shwartz et al . , it was mentioned that the ability of dfdba to induce bone formation is age dependant which can affect the ability of bone formation between various tissue banks with different donor selection criteria . in our study the mean cadaver ages were 35 years in all allografts , so there was no difference between them from this aspect . the graft materials used in this study is considered to be a xenograft , because it was human bone that was used in rabbit 's calvaria . one can supposed that this may have had a negative effect on the total bone formation , because studies pointed that allogenic bone grafts may be more effective than xenogenic ones.[2426 ] on the other hand it was stated that there is a homology between bmps from human , monkey , bovine , rabbit and rat extracellular - bone matrices and in a study by hollinger , et al . , the use of allogenic human bone in primates significantly increase new - bone formation in the csds . absence of statistically significant differences between allogenic bone materials can be attributed to the low number of study population , so the author recommended to compare different allografts in studies with the more sample size and in human - controlled trials . no difference was noted in bone formation and remained particles between three commercial bone allografts .

abstractbackgroundavailable literature on psoriasis and psoriatic arthritis ( psa ) demonstrates a tremendous burden of disease and suggests underdiagnosis and undertreatment.objectiveto obtain realworld physician perspectives on the impact of psoriasis and psa and its treatment on patients ' daily lives , including perceptions of , and satisfaction with , current therapies.methodsthe multinational assessment of psoriasis and psoriatic arthritis ( mapp ) surveyed dermatologists ( n = 391 ) and rheumatologists ( n = 390 ) in north america ( canada and the united states ) and europe ( france , germany , italy , spain and united kingdom).resultsdermatologists classified 20.3% and 25.7% of their patients as having severe psoriasis and severe psa respectively ; rheumatologists indicated that 48.4% of their psa patients had active disease . of the psoriasis patients complaining of joint pain , only 33.0% had a diagnosis of psa . an impact on daily activities or social / emotional wellbeing was recognized by 57.2% to 79.3% of physicians . in patients with moderatetosevere psoriasis , dermatologists reported 74.9% were receiving topical therapy , 19.5% conventional oral therapy and 19.6% biologics . dermatologists and rheumatologists reported similar rates of topical ( 45% ) and biologic ( 30% ) therapy utilization for their psa patients ; conventional oral therapy was more often prescribed by rheumatologists ( 63.4% ) vs. dermatologists ( 35.2% ) . reasons for not initiating or maintaining systemic therapies were related to concerns about longterm safety , tolerability , efficacy and costs ( biologics).conclusionphysicians in north america and europe caring for patients with psoriasis and psa acknowledge unmet treatment needs , largely concerning longterm safety / tolerability and efficacy of currently available therapies ; evidence suggests underdiagnosis of psa and undertreatment of psoriasis among dermatologists .

current published estimates of the worldwide prevalence of psoriasis range from approximately 13%.1 , 2 , 3 , 4 , 5 , 6 psoriatic arthritis ( psa ) occurs in approximately 1030% of psoriasis patients . in the vast majority of cases , joint involvement follows skin involvement , often by 10 years.7 , 8 patients with psoriasis and psa experience a significant burden of disease , including reduced quality of life , decreased physical function and increased comorbidities.7 , 9 , 10 available evidence suggests that numerous unmet needs exist in the care of these patients , including underdiagnosis2 , 7 and suboptimal treatment.11 , 12 , 13 available surveys offer insight into psoriasis and psa disease burden and treatment . however , most are limited to a specific geographic region , patients currently under the care of a physician and/or being treated in a specific healthcare setting and/or members of a patient association , or lack of both a patient and physician survey component . moreover , many surveys provide limited information on patient and physicianrelated factors influencing treatment decisions . to better understand the unmet needs from the patient and physician perspective , and to gain deeper insight into the impact of psoriasis and psa on patients ' lives , a populationbased survey of 4400 patients and 800 physicians in north america and europe the multinational assessment of psoriasis and psoriatic arthritis ( mapp ) survey is the firstofitskind , largescale , multinational survey conducted . the survey explores the diagnosis and impact of psoriasis and psa on healthrelated quality of life as well as attitudes towards current therapies . the mapp survey was developed with patient and physician input and aims to provide a better reflection of psoriasis and psa severity , including the impact of disease and its treatment on patients ' daily lives from the physician perspective . results from the multinational patient survey , published separately , identified unmet needs related to assessment of disease severity , psa screening and diagnosis and treatment options.8 the current report includes results from the multinational physician survey . the survey was conducted by abt srbi , inc ( new york , ny , usa ) . methodology and results of the patient survey have been reported.8 dermatologists and rheumatologists were identified through national databases for each country . physicians were contacted from this list through random sampling methods and offered participation in the survey . screening criteria for dermatologists required that 50% of their practice be devoted to medical dermatology . a total of 6530 dermatologists and 5445 rheumatologists were screened ; 391 dermatologists and 390 rheumatologists completed interviews . north american results combine findings from canada and the united states ; european results combine findings from france , germany , italy , spain and united kingdom . for this report , results reflect overall global findings , except when notable differences in responses were observed between north america and europe . the survey was conducted by abt srbi , inc ( new york , ny , usa ) . methodology and results of the patient survey have been reported.8 dermatologists and rheumatologists were identified through national databases for each country . physicians were contacted from this list through random sampling methods and offered participation in the survey . screening criteria for dermatologists required that 50% of their practice be devoted to medical dermatology . a total of 6530 dermatologists and 5445 rheumatologists were screened ; 391 dermatologists and 390 rheumatologists completed interviews . north american results combine findings from canada and the united states ; european results combine findings from france , germany , italy , spain and united kingdom . for this report , results reflect overall global findings , except when notable differences in responses were observed between north america and europe . dermatologists reported that 86.0% of office visits in a typical month were related to medical dermatology , with 16.1% specifically related to psoriasis and 4.1% to psa . overall , north american rheumatologists had twice as many psa patients as european rheumatologists . physician and practice demographics psa , psoriatic arthritis . psoriasis patients were most often referred to the participating dermatologist by a primary care physician ( 63.9% ) , another specialist ( 16.2% ) or another dermatologist ( 7.9% ) ; psa patients were most often referred by a primary care physician ( 51.2% ) , another specialist ( 21.0% ) or another dermatologist ( 7.0% ) . psa patients were most often referred to the participating rheumatologist by a primary care physician ( 51.7% ) , dermatologist ( 22.9% ) or another specialist ( 18.4% ) . dermatologists classified 20.3% of their psoriasis patients and 25.7% of their psa patients with severe disease , 38.1% and 38.3% with moderate and 40.5% and 32.9% with mild respectively . in psoriasis patients , dermatologists most commonly considered the location or size of skin lesions to be the most important factor contributing to disease severity ( fig . most commonly considered pain or swelling of the joints to be the most important factor contributing to disease severity ( fig . top five most important factors contributing to psoriasis ( a ) and psoriatic arthritis ( b ) disease severity . most dermatologists ( 62.6% ) reported that psoriasis impacted patients ' daily activities and/or work , and 79.3% reported it affected their patients socially and/or emotionally ; 92.1% agreed the disease burden of psoriasis is frequently underestimated . dermatologists recognized that a higher proportion of their psa patients ( 73.7% ) were impacted in their daily activities and/or work , and 76.8% were affected socially and/or emotionally . fewer rheumatologists recognized the effect of psa on daily activities and/or work ( 57.2% ) or its social and/or emotional toll ( 64.9% ) . however , most proactively discuss the effect of psa on daily activities ( 85.3% ) or its social and/or emotional toll ( 67.2% ) . dermatologists reported discussing the possibility of developing joint disease in all ( 43.5% ) or some ( 46.5% ) of their psoriasis patients . however , most dermatologists and rheumatologists ( > 75% ) acknowledged that psa is likely underdiagnosed because of a failure to connect skin and joint symptoms . according to dermatologists , 20.0% of their psoriasis patients complain of joint symptoms , with only 33.0% of these patients having a confirmed diagnosis of psa , which was most often made by another physician ( 48.7% ) or the participating dermatologist ( 39.1% ) . collaborative rheumatologist / dermatologist care for psa patients was reported by 75.6% of dermatologists and 42.9% of rheumatologists . dermatologists indicated they were solely responsible for prescribing decisions in 23.6% of their psa patients . in 30.8% , they were primarily responsible for prescribing decisions but patients were monitored by a rheumatologist for joint symptoms , and in 44.8% , rheumatologists made prescribing decisions but the responding dermatologist monitored skin symptoms . while rheumatologists indicated they were solely responsible for prescribing decisions in 66.1% of their psa patients , 34.2% might also be monitored by a dermatologist for skin symptoms and in 8.7% of patients , the dermatologist made primary prescribing decisions , whereas joint symptoms were monitored by the rheumatologist . table 2 summarizes physician estimates of the current treatment utilization by patients with moderatetosevere psoriasis or psa in their practice . dermatologists reported that 74.9% of their psoriasis patients were receiving topical therapy , 19.5% were receiving conventional oral diseasemodifying antirheumatic drug ( dmard ) therapy and 19.6% were receiving biologics . psa patients were more often receiving oral therapy when treated by rheumatologists ( 63.4% ) vs. dermatologists ( 35.2% ) , whereas dermatologists and rheumatologists both reported that approximately 30% of their psa patients were receiving biologics . current treatment utilization in patients with psoriasis and psa psa , psoriatic arthritis ; uvb / puva , ultraviolet b / psoralen+ultraviolet a. the top goal for dermatologists treating psoriasis patients was keeping signs / symptoms at bay ( 52.5% ) , followed by improving normal functional activities ( 22.3% ) and improving patient selfesteem ( 10.7% ) . dermatologists reported feeling optimistic ( 45.2% ) and/or hopeful ( 35.3% ) about managing a new patient with moderatetosevere psoriasis . however , approximately one in five felt that managing such a patient was timeconsuming ( 19.8% ) and complicated ( 18.6% ) . approximately , 60% of dermatologists felt that psoriasis patients require more time and support than their other patients and that the current structure of the healthcare system does not allow them adequate time for their care . dermatologists cited affordability and longterm safety risks with current medications as the greatest challenges in managing psoriasis patients ( fig . greatest challenge in managing psoriasis ( a ) and psoriatic arthritis ( b ) patients . when treating psa patients , dermatologists and rheumatologists reported the top goals were to reduce joint pain and stiffness ( 33.8% and 36.2% respectively ) , improve normal functional activities ( 28.4% and 23.6% ) and keep symptoms at bay ( 25.3% and 23.6% ) . dermatologists reported feeling optimistic ( 30.9% ) or hopeful ( 29.7% ) about managing psa patients with active disease , 26.5% felt it would be complicated and 15.4% said it was timeconsuming ; 17.4% thought they would need to refer to or involve other specialists . rheumatologists reported feeling optimistic ( 61.3% ) or hopeful ( 37.2% ) about managing a psa patient with active disease ; 29.5% agreed that psa patients require more time and support than their other patients . dermatologists most often cited differentiating psa from other arthritic diseases as the greatest challenge ( 24.6% ) , whereas rheumatologists most often cited delayed referral ( 31.0% ) ( fig . , 53.5% of dermatologists said they would prescribe topical therapies as monotherapy for their moderatetosevere psoriasis patients . dermatologists most commonly identified the primary challenge with topical therapies as lack of , partial or waning response ( 25.1% ) ; compliance ( 18.4% ) ; patient complaints of inconvenience / mess ( 18.2% ) ; and inability to cover the entire surface area ( 17.9% ) . dermatologists ( 47.6% ) most commonly felt they were comfortable , but conservative , with the use of conventional oral dmard therapy , or reported using it sparingly ( 20.5% ) , whereas most rheumatologists ( 68.5% ) reported that they felt conventional oral therapy provided benefit and used it broadly and only 9.0% used it sparingly . in addition , 18.7% of dermatologists and 16.1% of rheumatologists said patient contraindications were a main factor in preventing greater use of conventional oral therapies . rheumatologists commonly cited lack of response as a limitation to continuing therapy ( 35.4% ) . for dermatologists and rheumatologists who held back prescribing oral therapy because of patient refusal or concerns , the most common reasons were related to tolerability , longterm safety , lifestyle modifications and lack or loss of response ( fig . top five most commonly cited limitations for initiating conventional oral therapy ( a ) , continuing conventional oral therapy ( b ) and patient concerns regarding the use of conventional oral therapy ( c ) . approximately 40% of dermatologists and rheumatologists were comfortable , but conservative , in their use of biologic therapy . more rheumatologists ( 41.5% ) reported aggressive use compared with dermatologists ( 19.9% ) , and a greater proportion of dermatologists ( 10.5% ) reported not prescribing biologics compared with rheumatologists ( 1.8% ) . among dermatologists , 65.5% said they would initiate and manage the biologic therapy themselves , compared with 90.3% of rheumatologists . the main reasons for not initiating biologics were related to cost and longterm safety and tolerability concerns ( fig . 4a ) . rheumatologists commonly cited lack or loss of response as a limitation to continuing therapy ( fig . longterm safety was more commonly reported as a limitation to initiating biologics by dermatologists and rheumatologists from north america ( 32.6% and 54.5% respectively ) compared with europe ( 13.6% and 17.0% respectively ) . biologic therapyrelated tasks most commonly considered burdensome included prior authorization paperwork ; ensuring patients comply with laboratory results ; handling patient calls regarding questions or possible sideeffects ; teaching selfinjection ; and discussing with patients the use of and risks associated with injectable biologic therapy ( fig . top five most commonly cited limitations for initiating ( a ) and continuing ( b ) patients on biologic therapy and burdensome tasks / steps associated with biologic therapy ( c ) . while most dermatologists were somewhat or very satisfied with the effectiveness of conventional oral ( 84.2% ) or biologic therapy ( 95.9% ) , 27.8% and 25.6% were somewhat / very dissatisfied with their longterm safety , respectively , and 75.4% and 83.4% expressed concern about the health risks of their longterm use . likewise , most rheumatologists were somewhat / very satisfied with the efficacy of currently available therapies ( conventional oral therapy : 81.8% ; biologics : 97.9% ) . concern regarding the longterm safety of oral and biologic therapies was reported by 11.3% and 15.4% , of rheumatologists respectively . however , 53.6% and 68.5% of rheumatologists reported being somewhat / very concerned with the health risks of longterm use of oral or biologic therapies respectively . when prescribing topical therapy for psoriasis patients eligible for oral or biologic therapy , 7.9% of rheumatologists stated that their hesitation to prescribe systemic therapy was related to tolerability and longterm safety concerns . among the respondents , 53.0% of dermatologists and 30.5% of rheumatologists agreed that keeping a patient 's symptoms at bay was the best the patients could expect from conventional oral therapy ; 49.6% and 27.7% , respectively , felt that patients leaving their practice due to frustration or dissatisfaction with currently available therapies is an important issue ; and 26.3% and 13.3% , respectively , felt the treatment options could be worse than the condition itself . the most important attributes of an ideal therapy , identified by the respondents , were largely related to efficacy and safety , which generally mirrored what they considered the greatest unmet therapeutic needs ( table 3 ) . in addition , improved access to therapy and oral administration were among the five most commonly selected attributes ; another oral option and new mechanism of action were among the five most commonly noted unmet therapeutic needs . top five attributes of an ideal therapy and greatest unmet therapeutic needs psa , psoriatic arthritis . dermatologists classified 20.3% of their psoriasis patients and 25.7% of their psa patients with severe disease , 38.1% and 38.3% with moderate and 40.5% and 32.9% with mild respectively . in psoriasis patients , dermatologists most commonly considered the location or size of skin lesions to be the most important factor contributing to disease severity ( fig . most commonly considered pain or swelling of the joints to be the most important factor contributing to disease severity ( fig . top five most important factors contributing to psoriasis ( a ) and psoriatic arthritis ( b ) disease severity . most dermatologists ( 62.6% ) reported that psoriasis impacted patients ' daily activities and/or work , and 79.3% reported it affected their patients socially and/or emotionally ; 92.1% agreed the disease burden of psoriasis is frequently underestimated . dermatologists recognized that a higher proportion of their psa patients ( 73.7% ) were impacted in their daily activities and/or work , and 76.8% were affected socially and/or emotionally . fewer rheumatologists recognized the effect of psa on daily activities and/or work ( 57.2% ) or its social and/or emotional toll ( 64.9% ) . however , most proactively discuss the effect of psa on daily activities ( 85.3% ) or its social and/or emotional toll ( 67.2% ) . dermatologists reported discussing the possibility of developing joint disease in all ( 43.5% ) or some ( 46.5% ) of their psoriasis patients . however , most dermatologists and rheumatologists ( > 75% ) acknowledged that psa is likely underdiagnosed because of a failure to connect skin and joint symptoms . according to dermatologists , 20.0% of their psoriasis patients complain of joint symptoms , with only 33.0% of these patients having a confirmed diagnosis of psa , which was most often made by another physician ( 48.7% ) or the participating dermatologist ( 39.1% ) . collaborative rheumatologist / dermatologist care for psa patients was reported by 75.6% of dermatologists and 42.9% of rheumatologists . dermatologists indicated they were solely responsible for prescribing decisions in 23.6% of their psa patients . in 30.8% , they were primarily responsible for prescribing decisions but patients were monitored by a rheumatologist for joint symptoms , and in 44.8% , rheumatologists made prescribing decisions but the responding dermatologist monitored skin symptoms . while rheumatologists indicated they were solely responsible for prescribing decisions in 66.1% of their psa patients , 34.2% might also be monitored by a dermatologist for skin symptoms and in 8.7% of patients , the dermatologist made primary prescribing decisions , whereas joint symptoms were monitored by the rheumatologist . table 2 summarizes physician estimates of the current treatment utilization by patients with moderatetosevere psoriasis or psa in their practice . dermatologists reported that 74.9% of their psoriasis patients were receiving topical therapy , 19.5% were receiving conventional oral diseasemodifying antirheumatic drug ( dmard ) therapy and 19.6% were receiving biologics . psa patients were more often receiving oral therapy when treated by rheumatologists ( 63.4% ) vs. dermatologists ( 35.2% ) , whereas dermatologists and rheumatologists both reported that approximately 30% of their psa patients were receiving biologics . current treatment utilization in patients with psoriasis and psa psa , psoriatic arthritis ; uvb / puva , ultraviolet b / psoralen+ultraviolet a. the top goal for dermatologists treating psoriasis patients was keeping signs / symptoms at bay ( 52.5% ) , followed by improving normal functional activities ( 22.3% ) and improving patient selfesteem ( 10.7% ) . dermatologists reported feeling optimistic ( 45.2% ) and/or hopeful ( 35.3% ) about managing a new patient with moderatetosevere psoriasis . however , approximately one in five felt that managing such a patient was timeconsuming ( 19.8% ) and complicated ( 18.6% ) . approximately , 60% of dermatologists felt that psoriasis patients require more time and support than their other patients and that the current structure of the healthcare system does not allow them adequate time for their care . dermatologists cited affordability and longterm safety risks with current medications as the greatest challenges in managing psoriasis patients ( fig . . greatest challenge in managing psoriasis ( a ) and psoriatic arthritis ( b ) patients . when treating psa patients , dermatologists and rheumatologists reported the top goals were to reduce joint pain and stiffness ( 33.8% and 36.2% respectively ) , improve normal functional activities ( 28.4% and 23.6% ) and keep symptoms at bay ( 25.3% and 23.6% ) . dermatologists reported feeling optimistic ( 30.9% ) or hopeful ( 29.7% ) about managing psa patients with active disease , 26.5% felt it would be complicated and 15.4% said it was timeconsuming ; 17.4% thought they would need to refer to or involve other specialists . rheumatologists reported feeling optimistic ( 61.3% ) or hopeful ( 37.2% ) about managing a psa patient with active disease ; 29.5% agreed that psa patients require more time and support than their other patients . dermatologists most often cited differentiating psa from other arthritic diseases as the greatest challenge ( 24.6% ) , whereas rheumatologists most often cited delayed referral ( 31.0% ) ( fig . in all , 53.5% of dermatologists said they would prescribe topical therapies as monotherapy for their moderatetosevere psoriasis patients . dermatologists most commonly identified the primary challenge with topical therapies as lack of , partial or waning response ( 25.1% ) ; compliance ( 18.4% ) ; patient complaints of inconvenience / mess ( 18.2% ) ; and inability to cover the entire surface area ( 17.9% ) . dermatologists ( 47.6% ) most commonly felt they were comfortable , but conservative , with the use of conventional oral dmard therapy , or reported using it sparingly ( 20.5% ) , whereas most rheumatologists ( 68.5% ) reported that they felt conventional oral therapy provided benefit and used it broadly and only 9.0% used it sparingly . in addition , 18.7% of dermatologists and 16.1% of rheumatologists said patient contraindications were a main factor in preventing greater use of conventional oral therapies . rheumatologists commonly cited lack of response as a limitation to continuing therapy ( 35.4% ) . for dermatologists and rheumatologists who held back prescribing oral therapy because of patient refusal or concerns , the most common reasons were related to tolerability , longterm safety , lifestyle modifications and lack or loss of response ( fig . top five most commonly cited limitations for initiating conventional oral therapy ( a ) , continuing conventional oral therapy ( b ) and patient concerns regarding the use of conventional oral therapy ( c ) . approximately 40% of dermatologists and rheumatologists were comfortable , but conservative , in their use of biologic therapy . more rheumatologists ( 41.5% ) reported aggressive use compared with dermatologists ( 19.9% ) , and a greater proportion of dermatologists ( 10.5% ) reported not prescribing biologics compared with rheumatologists ( 1.8% ) . among dermatologists , 65.5% said they would initiate and manage the biologic therapy themselves , compared with 90.3% of rheumatologists . the main reasons for not initiating biologics were related to cost and longterm safety and tolerability concerns ( fig . 4a ) . rheumatologists commonly cited lack or loss of response as a limitation to continuing therapy ( fig . longterm safety was more commonly reported as a limitation to initiating biologics by dermatologists and rheumatologists from north america ( 32.6% and 54.5% respectively ) compared with europe ( 13.6% and 17.0% respectively ) . biologic therapyrelated tasks most commonly considered burdensome included prior authorization paperwork ; ensuring patients comply with laboratory results ; handling patient calls regarding questions or possible sideeffects ; teaching selfinjection ; and discussing with patients the use of and risks associated with injectable biologic therapy ( fig . top five most commonly cited limitations for initiating ( a ) and continuing ( b ) patients on biologic therapy and burdensome tasks / steps associated with biologic therapy ( c ) . while most dermatologists were somewhat or very satisfied with the effectiveness of conventional oral ( 84.2% ) or biologic therapy ( 95.9% ) , 27.8% and 25.6% were somewhat / very dissatisfied with their longterm safety , respectively , and 75.4% and 83.4% expressed concern about the health risks of their longterm use . likewise , most rheumatologists were somewhat / very satisfied with the efficacy of currently available therapies ( conventional oral therapy : 81.8% ; biologics : 97.9% ) . concern regarding the longterm safety of oral and biologic therapies was reported by 11.3% and 15.4% , of rheumatologists respectively . however , 53.6% and 68.5% of rheumatologists reported being somewhat / very concerned with the health risks of longterm use of oral or biologic therapies respectively . when prescribing topical therapy for psoriasis patients eligible for oral or biologic therapy , 7.9% of rheumatologists stated that their hesitation to prescribe systemic therapy was related to tolerability and longterm safety concerns . among the respondents , 53.0% of dermatologists and 30.5% of rheumatologists agreed that keeping a patient 's symptoms at bay was the best the patients could expect from conventional oral therapy ; 49.6% and 27.7% , respectively , felt that patients leaving their practice due to frustration or dissatisfaction with currently available therapies is an important issue ; and 26.3% and 13.3% , respectively , felt the treatment options could be worse than the condition itself . the most important attributes of an ideal therapy , identified by the respondents , were largely related to efficacy and safety , which generally mirrored what they considered the greatest unmet therapeutic needs ( table 3 ) . in addition , improved access to therapy and oral administration were among the five most commonly selected attributes ; another oral option and new mechanism of action were among the five most commonly noted unmet therapeutic needs . top five attributes of an ideal therapy and greatest unmet therapeutic needs psa , psoriatic arthritis . mapp is a unique largescale , multinational survey containing both a patient and physician component aimed at gaining a better understanding of global perspectives on the burden of psoriasis and psa and their treatment . results from the multinational patient survey showed a significant burden of disease and unmet treatment needs.8 we report the findings from the physician portion of the survey , which included dermatologists and rheumatologists who care for patients with psoriasis and/or psa . these findings largely support those of the patient survey , including a large disease burden , underdiagnosis of psa , differing perceptions of disease severity and challenges with current treatment options . while dermatologists and rheumatologists recognized the emotional and social toll that psoriasis and psa may place on patients , most acknowledged that the burden of disease is often underestimated . dermatologists estimated that 20% of their psoriasis patients and 26% of their psa patients had severe disease . in the patient portion of the mapp survey , slightly more psoriasis patients ( 27% ) and twice as many psa patients ( 53% ) rated their disease as severe.8 reasons for the disconnect may be related to the use of standardized tools by physicians vs. more subjective assessments by patients . in addition , patients and physicians were not matched , so results can not be directly compared . however , physicians and patients revealed notable differences in factors contributing to their assessment . while physicians reported that the most important factors contributing to disease severity were location and size of skin lesions in their psoriasis patients and pain and swelling of joints in their psa patients , itching and location of skin lesions were cited as important factors in patients ' assessment of disease severity.8 current findings show these factors , notably itching , 5a , b ) ; only 7% of dermatologists cited itching as the most important factor contributing to disease severity , compared with 38% of patients.8 itching is not captured in most currently utilized assessment tools , indicating a potential need to examine how disease severity is assessed . educating healthcare providers ( hcps ) regarding the importance of this outcome , as well as increased use of pruritus assessment tools in clinical studies , may help to bridge this disconnect . most important factors contributing to psoriasis disease severity , as reported by physicians and patients.8 most dermatologists and rheumatologists acknowledged that psa is likely underdiagnosed because of a failure to connect skin and joint symptoms ; however , dermatologists indicated they did not always discuss the possibility of psa with psoriasis patients . according to dermatologists , 20.0% of their psoriasis patients complained of joint symptoms , which is lower than the 44% of psoriasis patients in the mapp survey who indicated joint pain.8 in addition , 16% and 26% of patients with psoriasis alone reported symptoms possibly resembling enthesitis and dactylitis respectively.8 according to dermatologists , only 33.0% of psoriasis patients complaining of joint pain had a confirmed diagnosis of psa . consistent with previous reports,14 this may indicate that patients are not being fully assessed and diagnosed with psa despite the presence of joint symptoms , although joint pain may be attributable to a number of conditions other than psa . dermatologists most often cited differentiating psa from other rheumatologic or musculoskeletal diseases as the greatest challenge in managing psa , whereas rheumatologists most often cited delayed referral . educating dermatologists on the importance of discussing symptoms of pain , such as joint , back or foot pain , with psoriasis patients and when to refer patients to a rheumatologist may be helpful in increasing psa identification . the current findings further indicate that many patients with moderatetosevere disease may be undertreated , especially in the dermatology setting.15 only 39.1% of dermatologists reported their moderatetosevere psoriasis patients were receiving conventional oral ( 19.5% ) or biologic ( 19.6% ) therapy , and 54% indicated they would prescribe topical therapies as monotherapy for their moderatetosevere psoriasis patients . treatment rates in psa patients were higher , with approximately 65% of dermatologists reporting conventional oral ( 35.2% ) or biologic ( 30.6% ) use . the main reasons for not initiating or maintaining these therapies were related to concerns about longterm safety , tolerability and efficacy , as well as costs ( biologics ) . in addition , physician responses indicate that the most burdensome aspects of biologic therapy were related to the time requirements for patient education and management and prior authorization requirements . dermatologists and rheumatologists reported higher treatment rates compared with patients surveyed in mapp.8 this may be due to the fact that patients could participate in the mapp survey whether or not they were under the care of a physician , and may not have been treated because they were not seeing a physician for their disease . many dermatologists and rheumatologists felt that the management of psoriasis and psa was complicated , timeconsuming and challenging . approximately half of the dermatologists and one quarter of the rheumatologists felt that patients leaving their practice because of frustration or dissatisfaction with current therapies was an important issue . in the patient survey , many patients cited not seeing an hcp in the past 12 months because they did not think the hcp could help , and because of frustration , low expectations , and/or dissatisfaction with current therapies.8 furthermore , dermatologists and rheumatologists often perceive the management of psa as challenging . patients are typically seen by multiple hcps , and psoriasis and psa are associated with a number of comorbidities that can make treatment more complex.16 satisfaction with current therapies may impact patient management as well . patients generally expressed lower rates of satisfaction with current therapies compared with dermatologists and rheumatologists , particularly for ease of use and longterm safety.8 taken together , the findings of the patient and physician surveys suggest that available therapies may not be initiated because of longterm safety concerns , cost , need for injections and additional time requirements for therapies , and confirm the need for efficacious , less burdensome , costeffective therapies with improved safety and tolerability profiles . as with any survey , mapp was limited by factors such as accurate recall and interpretation of questions . because the survey was blinded , physicians could not be recontacted to clarify answers . the mapp survey lacked a control group and was not designed to capture differences in use of or attitudes towards various agents within a class of drugs or the impact of healthcare system requirements on decisionmaking . the findings of the physician portion of the mapp survey support the findings of the patient survey and highlight the importance of screening and assessing psoriasis patients for symptoms of psa , aligning patient and physician severity assessments and treatment goals , and the ongoing need for safe and effective therapies for psoriasis and psa .

electrical gradients are critical for many biological processes , including the normal function of excitable tissues , left - right patterning , organogenesis , and wound healing14 . the fundamental mechanisms that regulate the establishment and maintenance of such electrical polarities are poorly understood . using high - speed optical mapping of transmembrane potentials and calcium concentrations in the zebrafish heart , we have identified a gradient of electrical coupling across the developing ventricular myocardium . we excluded a role for cellular excitability , connexin localization , tissue geometry and mechanical inputs , but in contrast we were able to demonstrate that non - canonical wnt11 signals are required for the genesis of this myocardial electrical gradient . importantly , though the traditional planar cell polarity pathway is not involved , we obtained evidence that wnt11 acts to set up this gradient of electrical coupling through effects on transmembrane ca2 + conductance mediated via the l - type calcium channel . these data reveal a previously unrecognized role for wnt / ca2 + signaling in establishing an electrical gradient in the plane of developing cardiac epithelium through modulation of ion channel function . the regulation of cellular coupling through such mechanisms may be a general property of non - canonical wnt signals .

hearts , isolated from wildtype zebrafish embryos ( 24hpf to 72hpf ) were stained with the transmembrane potential - sensitive dye di-8-anepps ( invitrogen ) or the calcium - sensitive ratiometric dye fura-2 , am ( invitrogen ) for the measurements of action potentials and calcium transients , respectively . fluorescence intensities were recorded with a high - speed ccd camera ( redshirt imaging ) . propagation velocities of depolarizing waves were estimated as previously described , with some modifications ( see full description in the online methods ) . hearts , isolated from 72hpf zebrafish embryos were fixed in prefer fixative ( anatech ltd . ) and stained with primary antibodies : rabbit anti - connexin43 ( sigma ) 1:50 , mouse anti--catenin ( bd biosciences ) 1:200 , mouse anti - zn8 ( dshb ) 1:50 ; secondary antibodies : donkey anti - rabbit or mouse alexa - fluor-488 or -546 conjugated ( invitrogen ) 1:1000 . wnt11:cfp fusion protein was cloned downstream of cmlc2 promoter into the tol2kit expression system using gateway technology ( invitrogen ) . 10 g/l or 25 g/l of dna was co - injected with 25 g/l capped tol2 transposase mrna into 1 cell stage embryos . scientific ) was added to 30hpf old zebrafish embryos in egg water buffered with 5 mm hepes . isolated zebrafish hearts were incubated in a mix of thapsigargin 10 m ( sigma ) and caffeine 10 mm ( sigma ) in tyrode s solution for 45 min prior to optical imaging . hearts , isolated from wildtype zebrafish embryos ( 24hpf to 72hpf ) were stained with the transmembrane potential - sensitive dye di-8-anepps ( invitrogen ) or the calcium - sensitive ratiometric dye fura-2 , am ( invitrogen ) for the measurements of action potentials and calcium transients , respectively . fluorescence intensities were recorded with a high - speed ccd camera ( redshirt imaging ) . propagation velocities of depolarizing waves were estimated as previously described , with some modifications ( see full description in the online methods ) . hearts , isolated from 72hpf zebrafish embryos were fixed in prefer fixative ( anatech ltd . ) and stained with primary antibodies : rabbit anti - connexin43 ( sigma ) 1:50 , mouse anti--catenin ( bd biosciences ) 1:200 , mouse anti - zn8 ( dshb ) 1:50 ; secondary antibodies : donkey anti - rabbit or mouse alexa - fluor-488 or -546 conjugated ( invitrogen ) 1:1000 . wnt11:cfp fusion protein was cloned downstream of cmlc2 promoter into the tol2kit expression system using gateway technology ( invitrogen ) . 10 g/l or 25 g/l of dna was co - injected with 25 g/l capped tol2 transposase mrna into 1 cell stage embryos . scientific ) was added to 30hpf old zebrafish embryos in egg water buffered with 5 mm hepes . isolated zebrafish hearts were incubated in a mix of thapsigargin 10 m ( sigma ) and caffeine 10 mm ( sigma ) in tyrode s solution for 45 min prior to optical imaging .

hematopoietic stem cell transplantation ( hsct ) is widely used as a potentially curative treatment for patients with various hematological malignancies , bone marrow failure syndromes , and congenital immune deficiencies . the prevalence of oral complications in both autologous and allogeneic hsct recipients remains high , despite advances in transplant medicine and in supportive care . frequently encountered oral complications include mucositis , infections , oral dryness , taste changes , and graft versus host disease in allogeneic hsct . oral complications are associated with substantial morbidity and in some cases with increased mortality and may significantly affect quality of life , even many years after hsct . inflammatory processes are key in the pathobiology of most oral complications in hsct recipients . this review article will discuss frequently encountered oral complications associated with hsct focusing on the inflammatory pathways and inflammatory mediators involved in their pathogenesis .

hematopoietic stem cell transplantation ( hsct ) is a potentially curative treatment for patients with various hematological malignancies , bone marrow failure syndromes , and congenital immune deficiencies . stem cells can be obtained from bone marrow , peripheral blood , or umbilical cord blood . autologous hsct ( in which stem cells are derived from the patient ) is utilized to treat chemosensitive malignancies , such as multiple myeloma , non - hodgkin , and hodgkin lymphoma . its anticancer effect is entirely derived from the high - dose , myeloablative conditioning regimen , whereas a subsequent autologous stem cell infusion enables bone marrow recovery . allogeneic hsct ( in which the stem cells originate from a related or unrelated donor ) is often the preferred treatment in a number of other hematological malignancies such as acute and chronic leukemia and relapsed lymphoma , because of its graft versus leukemia / lymphoma ( gvl ) effect , which is an immunological response of donor - derived immune cells against malignant cells . in the late 1990s , a better understanding of gvl biology led to preparative regimens that involve less intensive conditioning radio - chemotherapy and are thus less directly toxic than myeloablative regimens . unlike traditional myeloablative conditioning , these reduced intensity conditioning ( ric ) regimens are primarily immunosuppressive to enable engraftment of the transplanted donor cells and depend on the graft to eradicate cancer . ric transplants can also be conducted in patients previously not eligible for myeloablative protocols , because of older age or medical condition . gvl responses are often accompanied by graft versus host disease ( gvhd ) , a complication of allogeneic hsct in which donor - derived immune cells including t- , b- , and natural killer ( nk ) cells raise an immune response against normal host tissue , such as the oropharynx , gut , skin , eyes , and liver . the overall prevalence of oral complications in patients receiving hsct is estimated to be 80% . frequently encountered acute oral complications include mucositis , local and systemic infections , oral dryness , and taste changes [ 36 ] . whereas in autologous hsct most of these problems have resolved after 6 months , patients that have been treated with allogeneic hsct may also later on experience complications associated with gvhd . inflammatory processes are the key in the pathobiology of most oral complications in hsct recipients . this review article will discuss frequently encountered oral complications associated with hsct focusing on the inflammatory pathways and inflammatory mediators involved in their pathogenesis . oral mucositis ( om ) is an inflammatory - driven process of the oral mucosa and is one of the best - studied oral side effects of cancer therapy . it is induced by radiation therapy and/or chemotherapy and is characterized clinically by mucosal damage ranging from mild inflammation presenting as erythematous atrophic lesions to extensive ulcerations penetrating the submucosa . in hsct recipients , mucositis is not limited to the oral cavity but may occur along the entire orodigestive tract . the mechanisms underpinning the pathobiology of mucositis are thought to be largely the same regardless of the location along this tract . the incidence of om has been estimated to range from 75% to 100% following myeloablative conditioning regimens and has been reported as the most painful and debilitating oral complication , significantly impairing quality of life ( qol ) . prospective studies reported that conditioning regimens containing high - dose melphalan , busulphan , and cyclophosphamide in combination with total body irradiation ( tbi ) were associated with severe om [ 911 ] . while om risk among patients receiving conditioning regimens including tbi exceeds 90% , the risk drops to 30%50% for individuals being treated with protocols without tbi . conditioning regimens are the most important parameters determining om risk , but patient - related factors are also involved , although the association is less clear . in particular the local tissue environment and mucosal responses to damaging stimuli , which may in part be genetically determined , govern the risk , course , and severity of mucosal injury [ 12 , 13 ] . genetic determinants of om risk include genes that regulate the availability of active chemotherapy drug metabolites . for example , evaluation of genetic variation in folate - metabolizing enzymes may help to identify patients at greater risk for methotrexate toxicity , but enzyme deficiencies may be relatively rare . in contrast , differences in the expression of genes associated with biological pathways that drive mucositis are more common . for instance , genetic polymorphisms associated with the expression of inflammatory mediators such as tumor necrosis factor- ( tnf- ) have been implicated in om risk in patients undergoing allogeneic hsct . recently , sonis et al . identified a single - nucleotide polymorphisms- ( snp- ) based bayesian network developed from saliva - sourced dna that may predict an individual 's risk to develop severe om following conditioning for autologous hsct . om following ric regimens is usually less severe and of shorter duration [ 15 , 16 ] . however , ric regimens may vary considerably in intensity and accompanying toxicity and more prospective studies are necessary . considerable progress has been made in the past years in understanding the pathobiology of mucositis [ 1719 ] and we will summarize recent insights . historically , om was viewed solely as an epithelium - mediated event that was the result of nonspecific toxic effects of radiation and/or chemotherapy on rapidly proliferating basal epithelial cells resulting in clonogenic cell death . this continues to be a component of a much more complex contemporary model of mucositis developed by sonis . this five - phase biological model describes a cascade of interrelated and overlapping genetic and histopathological events . these phases include initiation , upregulation / activation , signal amplification , ulceration , and healing involving epithelial and connective tissues of the mucosa ( figure 1 ) . the initiation phase in the pathobiology of mucositis is characterized by radio- and/or chemotherapy - induced dna and non - dna damage that results in injury of basal epithelial cells , submucosal cells , and endothelial cells . in particular submucosal cell death contributes to injury . in response to this damage , reactive oxygen species ( ros ) are generated , which amplify dna damage and clonogenic cell death and activate in the subsequent phase a number of transcription factors , including nuclear factor- ( nf- ) b [ 7 , 21 ] . nf-b is considered as the gatekeeper for various inflammatory pathways involved in mucositis . activation of nf-b occurs in virtually all epithelial and submucosal cells and induces expression of adhesion molecules and activation of the mitogen - activated protein kinase ( mapk ) and cyclooxygenase- ( cox- ) 2 pathways [ 19 , 21 , 22 ] . furthermore , the finding that nf-b activation can have both proapoptotic and antiapoptotic effects ( through apoptosis regulator bcl-2 genes ) makes it a significant factor in determining the fate of normal tissues following cytotoxic therapy . upregulation of nf-b generates the formation of interleukin- ( il- ) 1 , il-6 , and tnf- . these proinflammatory mediators stimulate additional injury through positive feedback loops ( signal amplification phase ) , whereby tnf- acts on pathways to reinforce nf-b activation . additional support for the role of cytokines has been provided by therapies that interfere with the development of mucositis by influencing the cytokine profile [ 2431 ] . together with alternative , nf-b independent pathways such as the ceramide pathway , this results in apoptosis of submucosal and basal epithelial cells leading to mucosal ulceration . furthermore , metalloproteinases ( mmps ) are involved in the pathobiology of mucositis [ 32 , 33 ] . damage to the submucosa is , besides from being a direct effect of radiation or chemotherapy , mediated by the activation of activating protein-1 ( ap-1 ) , which stimulates the secretion of mmps by fibroblasts [ 19 , 34 ] . tnf- is an important regulator of the transcriptional activity of ap-1 , by initiating the mapk signaling pathway activating c - jun amino - terminal kinase . moreover , il-1 and cox-2 may induce mmp activation . increased levels of mmp-2 , -3 , -9 , and -12 no significant correlation was found between levels of mmp-1 , mmp-8 ( neutrophil collagenase ) , and mmp-13 ( involved in degradation of extracellular matrix ( ecm ) and bone ) in oral rinsing samples and om scores in allogeneic hsct recipients . the ulcerative phase comprises loss of mucosal integrity and microbiological colonization with subsequent further proinflammatory cytokine production . it is associated with epithelial proliferation , often concurrent with hematopoietic recovery , reestablishment of local microbial flora , and absence of factors that interfere with wound healing such as infection and mechanical irritation . the ecm plays a significant role in signaling between tissues and is a complex structural network of fibrous proteins , proteoglycans , and glycoproteins . ecm stimulates epithelial cell migration , proliferation , and differentiation , leading to renewal of the mucosa . epidermal growth factor ( egf ) , transforming growth factor- ( tgf- ) , il-1 , and interferon- ( ifn- ) appear to promote this process by upregulating tgf- , which stimulate expression of fibronectin and collagen type iv . after healing , the oral mucosa appears normal , but appearances may be deceptive as ongoing angiogenesis and connective tissue maturation result in increased risk for om if additional cytotoxic therapy is administered . several anti - inflammatory cytokines and growth factors have been identified to have a protective effect . de koning et al . suggested a protective role of il-10 , after observing more severe intestinal damage following methotrexate treatment in il-10 deficient mice compared to wild - type controls . several animal studies indicated that il-11 , an inhibitor and downregulator of inflammatory mediators including nitric oxide ( no ) , reduces om . moreover , it was suggested that subcutaneous administration of il-11 reduced the severity of om by maintaining keratin production in epithelial cells , as well as by reducing mucosal proinflammatory cytokine expression . unfortunately , il-11 administration caused severe fluid retention and early mortality in a clinical trial , leading to early closure of the study . topical tgf-3 prior to chemotherapy administration negatively regulated epithelial cell proliferation and reduced om in a hamster model . it is mitogenic and promotes cell survival by upregulating bcl-2 genes , which suppress apoptosis . kgf-1 also activates nuclear factor erythroid 2-related factor 2 ( nrf2 ) that coordinates the expression of cytoprotective genes and upregulates il-13 , an anti - inflammatory cytokine that attenuates the effects of tnf- . human recombinant kgf-1 has been found beneficial for the prevention of om in patients treated with high - dose ct and tbi followed by autologous hsct [ 31 , 43 ] . it has been demonstrated in mice that perturbations of local immune responses to the microbiota can lead to spontaneous inflammation , and vice versa , and loss of microbial diversity is associated with a proinflammatory state [ 44 , 45 ] . shifts in the commensal oral bacterial flora associated with leukemia , neutropenia , direct effects from anticancer therapy , antibiotic use , hyposalivation , and mucosal surface changes are well documented [ 46 , 47 ] . for example , results from a study in breast cancer patients suggested a shift to a more complex oral bacterial profile following chemotherapy , and a recent study using 16s rrna and 454 pyrosequencing suggested that chemotherapy - induced changes of the bacterial composition were predictive for om risk . substitution with coagulase - negative staphylococci ( cons ) for streptococci was associated with om . the presence of porphyromonas gingivalis , a strictly anaerobic gram - negative microorganism associated with periodontitis , was shown to have a positive predictive value for mucosal ulcerations in hsct patients . it was suggested that p. gingivalis plays a role in the initiation of om by upregulating toll - like receptors ( tlrs ) , which facilitate nf-b activation . their effects are synergistic and in many cases reinforced by immune and/or inflammatory reactions of the oral mucosa . some defense proteins , like salivary immunoglobulins and heat shock proteins , are involved in both innate and acquired immunity . cationic peptides and other defense proteins ( e.g. , lysozyme , bactericidal permeability increasing protein ( bpi ) salivary amylase , cystatins , proline - rich proteins , mucins , peroxidases , and statherin ) are primarily involved in innate immunity . however , the role of saliva in the development of om is presently unclear [ 53 , 54 ] . once ulcerations are manifest , they represent a risk for systemic infection with bacteremia , fungemia , fever , and sepsis , particularly with concomitant neutropenia [ 7 , 5557 ] . the damaged mucosa may provide a portal of entry for microorganisms and inflammatory products into the bloodstream [ 5860 ] . om is the most likely origin of bacteremia with oral viridans streptococci most frequently resulting in fever and may lead to acute respiratory distress syndrome and septic shock [ 61 , 62 ] . bacteremia may also be caused by coagulase - negative staphylococci ( cons ) originating from the oral mucosa [ 50 , 63 ] . it should be noted , however , that , in only 30% of patients with mucositis and fever , a bacterial cause could be identified . mucosal barrier injury likely triggers a systemic inflammatory response , and this response by itself may cause neutropenic fever . infectious and inflammatory complications associated with ulcerative om may explain the observation reported in a number of studies that om is associated with an increased risk to early nontumor - related mortality in hsct recipients [ 6 , 64 , 65 ] . as described below tissue damage associated with mucositis is also thought to be involved in the pathogenesis of gvhd [ 66 , 67 ] . however , vokurka et al . , found no evidence for this notion in a study in which om was prevented by oral cryotherapy in allogeneic hsct recipients treated with high - dose melphalan conditioning regimens . in allogeneic hsct recipients , gvhd is frequently encountered and remains a major cause of morbidity and mortality . organs most at risk to be affected by this complex immunologic disorder include the skin , gastrointestinal tract including the oropharynx and liver , and eyes . ( i ) the graft must contain immunologically competent cells ( t cells ) ; ( ii ) the recipient must be incapable of rejecting the graft cells ; and ( iii ) the recipient must express tissue antigens that are not present in the donor [ 66 , 70 ] . the most important genetically defined proteins on host cells to which donor t cells respond are human leukocyte antigens ( hla ) . the degree of hla match of donor to patient is the most important risk factor for gvhd . additional risk factors include minor histocompatibility antigens ( mha ) , older age of patient , primary disease , graft source , donor parity and sex mismatch , the toxicity of conditioning regimens , and the effectiveness of gvhd prophylaxis [ 71 , 72 ] . once established , gvhd is difficult to treat . as gvhd and gvl responses usually come together , a too rigorous gvhd prophylaxis or treatment may carry the cost of increased risk of disease relapse . gvhd can be classified as either acute gvhd ( agvhd ) or chronic gvhd ( cgvhd ) , defined by clinical and pathologic features [ 74 , 75 ] . hyperacute oral agvhd may develop as early as one to two weeks after hsct , but the differential diagnosis of agvhd , om , and infection can be challenging . oral agvhd should be considered when infection has been excluded and ulcerations fail to heal with hematologic recovery 21 to 28 days after allogeneic hsct . the condition is potentially underrecognized , but it is estimated that between 35% and 60% of patients with agvhd have oral manifestations . a pilot study reported that patients undergoing ric hsct developed less oral agvhd than recipients of myeloablative allogeneic hsct . a recent observational study in allogeneic hsct following ric conditioning reported an incidence of oral agvhd of 7% . the most common sites involved at the initial diagnosis of cgvhd are skin ( 75% ) , mouth ( 51%63% ) , liver ( 29%51% ) , and eye ( 22%33% ) . however , the oral cavity may be the principal and sometimes the only site of involvement and can serve as a useful component of cgvhd diagnosis and staging . oral cgvhd may affect the mucosa and/or the salivary glands and may develop into mucosal sclerosis . although oral cgvhd is mild in the majority of patients , it should always be considered as clinically significant due to its often prolonged duration . in a subset of patients it is a continuous source of pain , impairing oral function , affecting alimentation and nutritional status , impeding the maintenance of oral health , and reducing quality of life [ 82 , 83 ] . in a cohort of ric hsct recipients , cgvhd - related oral symptoms developed with a median onset of seven months after transplant persisted for a median duration of six months and reoccurred in one - third of affected patients . chronic mucosal gvhd of the oropharynx is estimated to occur in 4583% of patients [ 76 , 84 ] and is characterized by lichenoid inflammation particularly affecting the tongue , buccal mucosa , and the lips ( figure 2 ) . clinical signs resemble those seen in lichen planus and include white hyperkeratotic reticulations and plaques , erythema , and ulcerations , which may be covered with a pseudomembrane . typical histopathological features include apoptotic bodies , satellite necrosis and lichenoid interface inflammation , and lymphocytic infiltration at the junction of the epithelium and subepithelial connective tissue [ 74 , 79 ] . it is important to be aware of other complications that can mimic oral cgvhd , including oral mucosal reactions to medications ( including mammalian target of rapamycin ( mtor ) inhibitors ) , local allergic reactions , infections , and second primary tumors . salivary gland dysfunction is common in hsct patients and is related to the conditioning regimen , dehydration , medication , and cgvhd . the primary symptom of salivary gland cgvhd is xerostomia ( subjective complaint of oral dryness ) , but patients may also describe oral sensitivity and burning . cgvhd of the salivary glands is probably underdiagnosed . some patients experience xerostomia induced by hsct conditioning ( particularly by tbi - containing regimens ) and anticholinergic medications , and this may persist through the period when salivary gland cgvhd develops , making onset and diagnosis less evident . salivary gland cgvhd mimics sjgren 's syndrome , which is commonly associated with xerophthalmia and may also relate to pulmonary gvhd involvement . saliva plays a critical role in mastication and swallowing , taste , speech , tooth remineralization , the maintenance of oral ph balance , and prevention of oral infections . patients with salivary gland cgvhd are at risk of developing complications because of diminished salivary defense mechanisms , including antifungal and anticariogenic activities [ 88 , 89 ] . castellarin and coworkers reported cgvhd - associated rapidly progressive dental caries with cervical and interproximal involvement . measurement of resting and stimulated whole - saliva flow rates , individual analysis of the risk of oral diseases , and preventive measures should be part of supportive care in these patients . mucoceles are subepithelial extravasations of sialomucin that occur at the epithelial - connective tissue interface around the obstructed duct of minor salivary glands . clinically it presents as a soft , fluid - filled elevation of the epithelium [ 75 , 91 ] . oral sclerotic involvement resulting in limited jaw opening can be the result of perioral and facial skin sclerosis , typically as an extension of generalized sclerotic changes . mucosal sclerosis is rare but may develop as a complication of long - standing severe ulcerative mucosal cgvhd . it may restrict jaw opening and tongue movement and may extend to the throat and esophagus resulting in dysphagia . it can be associated with pain and secondary ulceration , significantly impairing alimentation and performing oral hygiene measures . development of agvhd occurs in three overlapping and interrelated steps : ( i ) conditioning ; ( ii ) activation and expansion of alloreactive cells ; and ( iii ) the effector phase [ 74 , 92 ] . first , conditioning regimens induce tissue damage ( e.g. , mucositis and skin damage ) , inducing the release of adenosine triphosphate ( atp ) and other damage signals leading to secretion of tnf- , il-1 , il-6 , chemokines , and adhesion factors . this will promote activation and proliferation of host antigen presenting cells ( apcs ) , increased expression of costimulatory molecules , and migration of apcs to secondary lymphoid organs . the impact of the microbiota on gvhd has been recognized to be significant as it has been postulated that the intestinal microflora and endotoxin exert a crucial step in apc activation . translocation of bacteria and bacterial components through damaged epithelial barriers triggers additional production of inflammatory cytokines , particularly tnf- , il-1 , and il-12 . a strong correlation between increased tnf- serum levels and inflammation secondary to gvhd was reported to be associated with major shifts in the composition of the intestinal microbiota , which may aggravate the severity of inflammation . whether interactions between the oral microbiota and host defense mechanisms may contribute to the pathobiology of oral gvhd remains to be assessed . during the second phase activated host apcs encounter resting donor t cells and present host antigens to these cells , which become activated , following activation , t cells exit the lymphoid organs , enter the blood circulation , and subsequently migrate to the host target tissues ( e.g. , skin , orodigestive tract , and liver ) . in the third ( effector ) phase , a complex cascade of cellular mediators and inflammatory agents induce and amplify tissue damage [ 66 , 70 ] . donor t cells cause tissue damage via direct cytotoxicity against epithelial cells and release of interferon- ( ifn- ) and il-2 . this activates nk cells and resident macrophages that release proinflammatory cytokines including tnf- , il-1 , and il-6 leading to amplification of the proinflammatory cytokine cascade ( cytokine storm ) that is a hallmark of acute gvhd [ 101103 ] . a recent study presented an association between il1b polymorphisms and agvhd , as well as between il-1 levels , in both saliva and blood , and agvhd development . in addition , an association was found between the cc genotype and high levels of il-1 suggesting that assessing the kinetics of il-1 in both fluid types may be useful for monitoring the progression of the disease . depending on the conditions of activation and subsequent cytokine profile , cd4 + cells can be subdivided into th1 , th2 , th17 , and other subtypes . although oversimplified , th1 , th2 , and th17 can be characterized by secretion of ifn- , il-4 , and il-17 , respectively . activation of cd8 + cytotoxic t cells is accompanied by increased production of effector molecules , which perpetuate the damage . both fas / fasl - dependent and perforin / granzyme - dependent apoptosis are important in gvhd - induced tissue damage . traditionally , agvhd is considered to be driven by th1 cytokines and mediated by cd8 + effectors . however , the pathobiology appears far more complex and b cells may also play a role in the development of agvhd . our understanding of cgvhd is limited , partly because of the difficulty with generating animal models that are true representatives of clinical disease . the first theory is end - stage alloreactivity , in which donor t cells augment a th2 immune response . the second theory postulates that cgvhd is due to poor immunologic recovery with the development of autoreactive t lymphocytes due to lack of thymic control or peripheral mechanisms of deletion . likely , the pathogenesis of cgvhd begins with uncontrolled expansion of donor t cells in response to both allo- and autoantigens . the activated t cells will subsequently cause target organ damage by inflammatory cytokines , cytolytic attack , and fibrosis and/or by promoting b - cell activation and production of autoantibodies . evidence exists that b - cell deregulation and the expansion of host b cells contribute to cgvhd . miklos et al . demonstrated that the presence of antibodies directed to h - y proteins ( a transplantation antigen that can lead to rejection of male grafts by female recipients ) correlates with cgvhd . also , elevated levels of b - cell activating factor ( baff ) contribute to b - cell activation in patients with active cgvhd . furthermore , young et al . described that donor b cells are activated by donor cd4 + t cells to upregulate mhc ii and costimulatory molecules . acting as efficient apcs , activated donor b cells enhance donor cd4 + t clonal expansion , thereby augmenting the capacity of these cells to induce autoimmune - like cgvhd . indeed , comparable to autoimmune diseases , both t- and b - cell responses appear to play a role in the pathogenesis of cgvhd , suggesting that this reflects a general loss of tolerance including abnormalities in the function of regulatory ( treg ) cells . impairment of tregs is associated with loss of peripheral tolerance and with development of cgvhd . acute gvhd has long been conceived to be dependent upon type i cytokine - driven cd8 effectors , whereas cgvhd has been associated with type ii cd4 t cells and th2 cytokines ( il-4 and il-10 ) are considered to be the predominant cytokines in the pathobiology of cgvhd . however , the mechanisms are far more complex , as both agvhd and cgvhd are initially characterized by increased th2 cytokines production . studies have also shown decreased th2-type cytokine levels among patients who developed cgvhd compared with those who did not [ 139 , 140 ] . taken together , evidence of th2 involvement in cgvhd is limited and , depending upon the animal model used , all 3 major subtypes of cd4 cells ( th1 , th2 , and th17 ) have been implicated in cgvhd . imanguli and coworkers systematically examined oral mucosal biopsies and assessed that the clinical severity of oral cgvhd was correlated with the presence of apoptotic epithelial cells . these cells were often found adjacent to infiltrating effector - memory t cells , expressing markers of cytotoxicity and type i cytokine polarization ( t - bet+ t cells ) . accumulation of t - bet+ t - cell effectors was associated with increased proliferation and the expression of the type i chemokine receptor cxcr3 . in both infiltrating cells and keratinocytes , increased expression of the cxcr3 ligand mig ( cxcl9 ) and il-15 , ifn - inducible factors , type i differentiation , and expansion of alloreactive effectors was observed . salivary gland cgvhd is characterized by typical histopathological changes ( figure 3 ) including periductal mononuclear infiltration particularly of cd45 , cd45ro , cd4 and cd8 positive cells , atrophy of salivary gland lobules , and periglandular fibrosis [ 86 , 143 , 144 ] . infections , including those from oral sources , are a frequent complication of hsct [ 70 , 76 , 92 ] . risk factors for obtaining infections include the underlying malignant disease , the medical condition and comorbidities , the presence of chronic or latent infections , the type of transplant , the source of stem cells , the use of antimicrobials , mucosal barrier loss , immunosuppression and myelosuppression induced by hsct conditioning , and gvhd and/or gvhd management . one depends on nonspecific defenses such as the integrity of surface barriers and presence of systemic or salivary antimicrobial agents , such as defensins . the other major defense against infections is the immune system , of which virtually all components are deficient after hsct or suppressed by immunosuppressive therapy to prevent gvhd . uncomplicated recovery starts with healing of the mucosal tissues and recovery of granulocytes and nk cells about two weeks after myeloablative conditioning . however , t - cell and b - cell immune responses against viral , bacterial , and fungal organisms may be suppressed for a prolonged period of time , particularly in the setting of gvhd . oral infections may be associated with a wide variety of microorganisms , including bacteria , fungi , and viruses , virtually all of which may give rise to systemic infectious complications in the transplant population . the time of occurrence and appearance of the lesions may contribute to the differential diagnosis . however , coexistent oral conditions such as om and gvhd often complicate adequate and prompt diagnosis of opportunistic infections . as described previously , the oral bacterial flora changes before and following chemotherapy . in a cohort of 37 allogeneic hsct recipients a significant increase of oral colonization with potentially pathogenic microorganisms ( predominantly enterococcus faecalis and candida spp . ) it is important to be aware of modified clinical symptomatology of bacterial infections during the neutropenic phase of hsct . erythema , pain , edema , and fever may be the only clinical signs of an otherwise purulent infection . om is acknowledged to be the principal risk factor for bacteremia due to oral viridans streptococci ( ovs ) , but bacteremia with cons may also originate from the oral cavity . in addition to infections related to the oral mucosa , chronic infections associated with the dentition may give rise to complications . periodontal infections , in particular , may represent an easily overlooked source of bacteremia and systemic infection in neutropenic patients [ 150 , 151 ] . periodontal infection and inflammation may contribute to the risk of developing ovs and cons bacteremia during neutropenia following hsct . moreover , the reported positive association between p. gingivalis and om suggests at least a mucosal reaction to this bacterial challenge . there is anecdotal evidence that dental infection and inflammation also contribute to oral gvhd . candidiasis is typically caused by opportunistic overgrowth of c. albicans , a commensal oral yeast . it may be associated with a dry mouth , taste disturbances , and mucosal discomfort [ 153 , 154 ] . several variables contribute to its clinical expression , including immunosuppression , mucosal injury , and salivary dysfunction . in addition , antibiotics may alter the oral flora , thereby creating a favorable environment for fungal overgrowth . the most common forms of intraoral candidiasis reported in oncology patients are pseudomembranous and erythematous candidiasis . current prophylactic strategies have reduced systemic candidiasis , although oropharyngeal and esophageal infections remain a common complication with potentially serious consequences . it has been recognized that different candida species provoke different immunologic reactions , making some strains more virulent than others . during oral infection with candida , villar et al . demonstrated that highly invasive strains of c. albicans triggered higher levels of proinflammatory cytokines , including il-1 , il-6 , il-8 , and tnf- in epithelial cells and il-6 , il-8 , monocyte chemotactic protein- ( mcp- ) 1 , mcp-2 , and granulocyte colony - stimulating factor ( gcsf ) in endothelial cells . c. glabrata was associated with oral ulcerations in hsct recipients , as described by laheij et al . . also identified c. kefyr as having a positive predictive value for mucosal ulcerations , but its prevalence is unknown in larger populations . did not report a positive correlation between candida colonization and the presence or severity of om in hsct patients . these results indicate that the role of candida species in the pathogenesis of om remains to be elucidated in more detail . interestingly , van der velden et al . suggested that the mycobiome has a role in the pathogenesis of agvhd . colonization of the mucosa may trigger intestinal agvhd , potentially by the induction of th17/il-23 responses through activation of pattern recognition receptors by fungal motifs . whether this also may occur in the oral cavity noncandidal fungal organisms may be also associated with oral infection in immunocompromised cancer patients , including infection by species of aspergillus , mucormycosis , and rhizopus . lesions associated with these fungi may resemble om lesions but more detailed microbiologic documentation is needed . moreover , viruses may give rise to systemic infectious complications and may trigger gvhd [ 167 , 168 ] . most often , herpes simplex virus ( hsv ) , varicella - zoster virus ( vzv ) , and epstein - barr virus ( ebv ) infections result from reactivation of latent virus , while cytomegalovirus ( cmv ) infections can result from either virus reactivation or via a newly acquired virus . this reduces viral shedding and herpetic lesions , although reactivation is still possible in patients receiving prophylaxis . when reactivation of hsv-1 occurs , ulcers may develop and aggravate om , or they may be confused with this condition . rping et al . studied the association of il-1 and tnf- in oral saliva and viral pathogens with the severity of ulcerations in autologous hsct . similarly van der beek et al . reported that oral shedding of hsv-1 predicts ulcerations . however , in these studies patients did not receive antiviral prophylaxis , whereas in hsct recipients getting acyclovir prophylaxis hsv seems not a major etiologic agent of ulcerative om . like hsv-1 and -2 , vzv is latent in neurons , but viral particles are transported via axons to mucosa where the virus continues replicating in epithelial cells . increased risk of vzv reactivation extends from three to twelve months after transplant , with allogeneic transplant recipients being at the highest risk . salivary glands seem an important reservoir of cmv , as the virus can be shed in saliva and infected saliva is the main mechanism for transmitting cmv . correia - silva et al . found a positive correlation between cmv dna loads in saliva and blood in allogeneic hsct recipients . although a recent study did not find a correlation between cmv in oral rinsing samples and oral ulcerations , oral ulceration due to cmv and cases of coinfection of hsv and cmv in oral ulcers have been reported . cmv infection causes monocyte and t - cell activation and activation of the tnf system , concomitant with an increase in plasma levels of il-10 . moreover , cmv may modulate immunological status of host cells by inducing local production of proinflammatory cytokines such as tnf , il-1 , il-6 , il-8 , and il-10 [ 180 , 181 ] . liu et al . investigated the mechanism of cmv - specific t - cell immune responses after hsct in cmv - specific cd8 + t cells . they could not define the critical threshold or absolute number of cmv - specific cd8 + t cells that protect against cmv reactivation . hsct recipients may be at risk of developing ebv - related lymphomas and ebv - associated carcinomas of the head and neck region . posttransplantation lymphoproliferative disorder ( ptld ) is the most common malignancy in the first year following allogeneic hsct . oral involvement of ptld is rare ; it may manifest as a crater - like gingival defect or an ulcerated dark - red mass . in addition , oral hairy leukoplakia has been attributed to ebv infection in hsct recipients . oral lesions caused by nonherpes viruses including adenovirus and human papilloma virus ( hpv ) have been described . a case of rapidly enlarging , biopsy - documented oral verruca vulgaris in a patient undergoing myeloablative hsct has been reported . taste dysfunction ( dysgeusia ) negatively influences qol and may lead to impaired nutrition and weight loss . while conditioning regimen - related dysgeusia is typically associated with the onset of om and resolves one to two months following hsct , taste disorders may persist or develop de novo in allogeneic hsct recipients . patients may report a rapid decrease in their sense of taste that is temporally associated with the onset or exacerbation of cgvhd , suggesting that the epithelial - derived taste receptor cell is an immune - based target . antibiotics may have a negative impact on taste , whereas drugs used to prevent and treat gvhd ( e.g. , cyclosporine , mtor inhibitors ) can also induce neurological changes that result in altered taste . with increased numbers of survivors , late effects of hsct and concomitant therapies have become of increasing importance . hyposalivation may persist when associated with salivary gland cgvhd , putting patients at risk for rapidly progressing dental demineralization and caries . in addition , disturbances in tooth formation and jaw growth and development may be present in pediatric hsct survivors . among these late effects , second malignancies have been recognized , including ptld , hematologic malignancies , and solid tumors . solid tumors may develop many years after hsct . in the vast majority of cases , oral tumors are squamous cell carcinomas ( scc ) . several authors have described cases of scc at oral and skin sites previously affected by cgvhd - related inflammatory processes , suggesting that cgvhd is a risk factor . in addition , prolonged immunosuppressive therapy may contribute to scc risk . in a large retrospective study , hsct recipients had a 20- to 30-fold risk for oral cancer 10 years or more after allogeneic hsct . changes in the oral mucosa due to gvhd can make early detection and diagnosis of scc clinically challenging . long - term follow - up of hsct patients is recommended to detect cancers at an early stage , and patients should be informed of cancer risk and educated to avoid life styles that can potentiate the risk of developing oral scc . . a better understanding of the pathobiology of these complications and risk factors is necessary in order to develop more efficient preventative and therapeutic strategies . hsct conditioning regimens are important parameters determining om risk , but patients differ considerably in their susceptibility to develop severe om . exciting progress has been made in predicting an individual 's genetic risk for severe om following conditioning for autologous hsct by sonis et al . , who recently identified a snp - based bayesian network developed from saliva - sourced dna . studies in larger number of hsct recipients may ultimately result in a clinically useful saliva - based tool predicting severe om . in addition , studies evaluating the role of the oral microbiome in maintaining oral homeostasis versus aggravating om are promising . recently it has been suggested that cancer regimen related adverse events do not occur in isolation but rather develop in clusters . toxicities may be related with regard to the time of their development only , but there may be also causal relationships in which one toxicity predisposes to a subsequent complication . moreover , toxicities of cancer treatment may have a common pathobiological background and share genetically determined and other risk factors . as discussed above , inflammation , particularly the upregulation of proinflammatory cytokine pathways , is a major common driver of complications in hsct recipients and interventions blocking these pathways may affect multiple complications . oral complications that seem to be interrelated include om , hyposalivation , taste alterations , oral infections , and oral gvhd , although in the past most studies have focused on isolated toxicities , and more research is necessary to assess the nature of relationships . oral complications also link to complications involving nonoral tissues as well as those manifesting systemically . for example , in neutropenic hsct recipients om is associated with bacteremia , fever , and sepsis . such associations may , at least in part , explain the observation that om is associated with an increased risk to early nontumor related mortality in hsct recipients [ 6 , 64 , 65 ] . in addition to having a genetic predisposition for ( multiple ) inflammatory complications of hsct , the presence of inflammation may lead to a dysregulated and exaggerated inflammatory response following a subsequent inflammatory stimulus . interestingly , a recent study reported that , at the time of diagnosis of acute leukemia , high prechemotherapy plasma levels of both pro- and anti - inflammatory cytokines and low levels of an antimicrobial protein pro - ll-37 were associated with the highest om risk , suggesting that a proinflammatory state preceding high - dose chemotherapy may predispose to complications . similarly , it has been proposed that periodontitis ( present before the initiation of cancer treatment ) may coinduce an exaggerated inflammatory response following ( chemo)radiation in patients with head and neck cancer , leading to more severe om . it is well established that periodontal disease induces low - grade systemic inflammation characterized by increased levels of proinflammatory cytokines and acute phase proteins in peripheral blood ( reviewed by van dyke and winkelhoff , 2013 ) . this is the result of bacteria or bacterial and inflammatory products translocating through inflamed and ulcerated pocket epithelium into the circulation . periodontitis and om may be associated with a primed inflammatory response as proposed by the two - hit model . the model has previously been used to explain the pathogenesis of acute respiratory distress syndrome ( ards ) following cardiopulmonary bypass and the association between periodontitis and inflammation driven systemic diseases including rheumatoid arthritis [ 200 , 201 ] . likewise , it can be hypothesized that oral infections including periodontitis may predispose to om in hsct recipients . and taking this concept a step further , it may be speculated that inflammatory conditions ( oral or elsewhere ) either present before cytotoxic therapy , or developing as a result of therapy may predispose to other inflammation - driven acute or late complications in hsct recipients . for example , there is anecdotal evidence that periodontal inflammation triggers oral gvhd , whereas gvhd ameliorates when periodontal or other foci of infection are eliminated . likewise periodontitis and/or om may be involved in sepsis risk even in the absence of bacteremia . although a relationship between gastrointestinal mucositis , fever , and increased levels of inflammatory markers in the absence of bacteremia has been proposed [ 57 , 202 ] , the relative contribution of om to systemic inflammation remains to be investigated . to explore these hypotheses , longitudinal observational studies involving large numbers of patients looking into associations of oral and nonoral complications and their potentially common underlying mechanisms furthermore , investigations using novel techniques will provide an opportunity to evaluate the role of the oral and gastrointestinal microbiome in mucositis and gvhd . together with studies aimed at assessing the relationship between the microbiome and host factors including epithelial defense mechanisms , this may lead to successful strategies to prevent or ameliorate these complications . moreover , longitudinal studies using salivary samples may identify genetic risk factors for om as well as for other regimen related complications . in addition , studies may identify salivary biomarkers that may predict or facilitate diagnosis of om and gvhd or their response to therapy . in conclusion , a more holistic approach that includes clinical and translational studies on oral as well as nonoral complications of hsct may lead to a better understanding of potential commonalities between complications and may open new avenues for prevention and treatment .

backgroundlung perfusion is one of the key components of oxygenation . it is hampered in pulmonary arterial diseases and secondary due to parenchymal diseases.methodsassessment is frequently required during the workup of a patient for either of these disease categories.resultsthis review provides insight into imaging techniques , qualitative and quantitative evaluation , and focuses on clinical application of mr perfusion.conclusionthe two major techniques , non - contrast - enhanced ( arterial spin labeling ) and contrast - enhanced perfusion techniques , are discussed .

pulmonary perfusion is altered in physiological and pathological conditions such as pulmonary embolism , pulmonary hypertension and chronic obstructive disease . therefore , assessment of the pulmonary perfusion is of physiological and clinical importance . as the lung is a large , diffuse and inhomogeneous organ it one of these techniques is magnetic resonance imaging ( mri ) . for more than 20 years mri two principal techniques are established : arterial spin - labeling and contrast - enhanced perfusion . especially the tremendous technical developments of the recents years regarding hardware and new sequences make mr - based perfusion imaging broadly available . therefore , this review article will elucidate the current state of the art sequences , clinical applications and future developments of each technique . mr offers the possibility to mark a specific part of spins magnetically by selective radiofrequency ( rf ) excitation , leading to a specific magnetisation to a selected fraction of blood . this technique is called arterial spin labeling ( asl ) and requires no intravenous application of contrast material and is therefore considered to be completely non invasive . it has the advantage that measurements can be made repeatedly over very short time periods ( seconds ) . the absence of contrast also means that the measurements can be repeated indefinitely as it has not to be cleared out from the blood before reimaging is possible ( also , the absolute number of measurements is limited by the total amount of contrast material that can be applied in 1 day ) . there are some limitations of these techniques : ( 1 ) more difficult to implement compared with dynamic contrast - enhanced imaging ; ( 2 ) requires a regular cardiac cycle ; ( 3 ) single slice technique and 58 slices have to be acquired to cover the whole lung ; ( 4 ) the limited voxel resolution ( i.e. 5 5 30 mm ) . in essence , two ecg - gated images of a selected slice are taken 58 s apart during a single apnea . the two images differ only in the way that tagging radiofrequency ( rf ) pulses change the signal of blood flowing into the imaged section while keeping the signal from the stationary structures unchanged between images . subtraction of the two images leaves an image in which the signal from a volume element ( voxel ) is proportional to the amount of pulmonary arterial blood delivered during the previous heart cycle [ 3 , 511 ] . in principle true fast imaging with steady precession ( true fisp ) sequences in particular has been found to be ideally suited for assessment of pulmonary perfusion . specific types of asl techniques that allow for quantification of regional pulmonary perfusion during a single breath hold are called flow - sensitive alternating inversion recovery ( fair ) and flow - sensitive alternating inversion recovery with an extra radio frequency ( rf ) pulse ( fairer ) . perfusion - weighted images are obtained from the subtraction of a control and tag images , which are interleaved and can be acquired within one breathholding period of 2027 s ( for each slice ) . cardiac motion and pulsatile flow of the descending aorta can generate smearing and flow artefacts , respectively . spatial variation between the control images can lead to ghosting of the blood vessels , and spatial movement between the control and tag images causes the appearance of the adjacent dark - bright pairs of the blood vessels . a closely related technique , called the t1 method , has also been used to measure pulmonary perfusion ( fig . 1 ) [ 14 , 15 ] . it differs primarily in how the experiment is conceptualised as the delivery of a bolus of tagged spins or as an apparent alteration of the longitudinal relaxation time . perfusion - weighted images are acquired in a single shot by completely saturating the static background tissue before image acquisition . the decrease of the lung t1 values in the same slice position from the global to the selective experiment is statistically significant and results from the wash - in effect of the noninverted blood water h spins outside the imaging slice . because the perfusion - weighted image is acquired in a single shot , artefacts arising from patient motion or different respiratory levels can be avoided . furthermore , only very short acquisition times ( 5 s per slice ) are required for the perfusion - weighted image , facilitating rapid and comfortable examinations . this is especially important for lung imaging , as some patients are not able to hold their breath for more than a few seconds.fig . 1arterial spin labeling ( asl ) image in a healthy volunteer ( sagittal orientation , right lung ) . the spatial resolution is 3.9 3.9 10 mm , which allows for visualisation of the major and minor fissure . on the other hand the signal - to - noise ratio is low , making an detailed evaluation difficult arterial spin labeling ( asl ) image in a healthy volunteer ( sagittal orientation , right lung ) . the spatial resolution is 3.9 3.9 10 mm , which allows for visualisation of the major and minor fissure . on the other hand the signal - to - noise ratio is low , making an detailed evaluation difficult asl - based techniques allow for calculation of relative pulmonary blood flow ( rpbf ) in ml/100 g / min [ 12 , 16 , 17 ] . for conversion of rpbf into pbf the lung density is required , which is usually assumed to be constant with 0.33 g / ml ( at functional residual capacity ) . however normal values are reported to be in the range of 5.5 ml / g / min . using a rapid ir snapshot flash technique , a spin labeling method within the imaging slice was used in combination with a simple two - compartment lung tissue model . the measured pulmonary perfusion rates of the healthy lung parenchyma were on the order of 400 to 600 ml/100 g / min , depending on the subject investigated and the actual slice position in the lung . in healthy volunteers ( n = 6 ) a quantitative comparison between non contrast - enhanced and contrast - enhanced techniques was performed . two coronal slices ( middle and dorsal part of the lung ) were assessed and maps of relative pulmonary blood flow ( rpbf ) were calculated no absolute perfusion measures were calculated . by placing ten regions of interest in each lung , a right - to - left perfusion ratio analysis was performed . for the dorsal slice the middle position however showed a 1643% underestimation of fair - derived rpbf in the right lung . this was explained by tracer saturation for the right lung as the right pulmonary artery is in the plane of both pulses needed for tagging and readout . in an internal control this difference was not present if slices were acquired in a sagittal orientation . based on the strength and limitations noted above , the strength of the techniques lies in the ability to study physiological aspects of the lung perfusion on a regional level [ 2124 ] . important for all perfusion studies is to notice that the snr of asl images is low , particularly in the anterior portions of the lung . therefore , positioning the patient so that the area of interest is down - gravity may increase snr and improve visibility of perfusion defects . also , signal intensity and subsequently perfusion increases during examination on end - expiratory breath - hold compared to end - inspiratory breath - hold [ 25 , 26 ] . normal aging is associated with a decline in pulmonary function and efficiency of gas exchange . the effect of aging on spatial heterogeneity of pulmonary perfusion was studied on 56 healthy , non smoking volunteers with an age range of 2176 years . relative dispersion increased significantly with increasing age by 0.1/decade until age 5059 years , and a significant positive relationship between relative dispersion and age was found . therefore , it was not possible to investigate if the age - related parenchymal changes were the cause for this phenomenon . in an early study keilholz et al . demonstrated the ability of asl to depict the gravity - dependent perfusion effects . healthy volunteers were placed inside the scanner on their right and left lateral side and coronal images were acquired within a 10-s breath - hold using a fairer haste sequence . in both positions , an increase in the intensity of the dependent lung was found ( 229% for left lateral , 40% for right lateral ) . a pulmonary emboli model was introduced using a balloon catheter that was placed in the left pulmonary artery . after occlusion of the left pulmonary artery a set of fairer images followed by a contrast - enhanced angiography was performed . the huge perfusion defect ( whole left lung ) it has to be noted again , that 8 s were required for a single slice of asl , while the 3d acquisition of the contrast - enhanced perfusion needed 30 s. a similar experiment was done in 5 rabbits were asl perfusion and ventilation images using hyperpolarised 3helium were assessed . after occlusion of the pulmonary artery by a balloon catheter the ventilation was unchanged and the asl perfusion images demonstrated the large perfusion defect . in patients susceptible to high altitude pulmonary oedema ( hape ) it was possible to demonstrate the uneven hypoxic pulmonary vasoconstriction compared to non - susceptible patients by assessing the relative dispersion of blood flow . up to now , only few reports exist on the clinical application of non contrast - enhanced perfusion techniques . due to the lengthy acquisition times and relatively low signal increase , it may be the imaging technique of choice for pregnant women to rule out pulmonary emboli . however , in this stetting other native imaging techniques , like steady - state free precession sequences , also showed a good clinical potential . therefore , the main application will be in exploration of physiological processes where the possibility of unrestricted measurements outweighs the prolonged acquisition time . mr offers the possibility to mark a specific part of spins magnetically by selective radiofrequency ( rf ) excitation , leading to a specific magnetisation to a selected fraction of blood . this technique is called arterial spin labeling ( asl ) and requires no intravenous application of contrast material and is therefore considered to be completely non invasive . it has the advantage that measurements can be made repeatedly over very short time periods ( seconds ) . the absence of contrast also means that the measurements can be repeated indefinitely as it has not to be cleared out from the blood before reimaging is possible ( also , the absolute number of measurements is limited by the total amount of contrast material that can be applied in 1 day ) . there are some limitations of these techniques : ( 1 ) more difficult to implement compared with dynamic contrast - enhanced imaging ; ( 2 ) requires a regular cardiac cycle ; ( 3 ) single slice technique and 58 slices have to be acquired to cover the whole lung ; ( 4 ) the limited voxel resolution ( i.e. 5 5 30 mm ) . in essence , two ecg - gated images of a selected slice are taken 58 s apart during a single apnea . the two images differ only in the way that tagging radiofrequency ( rf ) pulses change the signal of blood flowing into the imaged section while keeping the signal from the stationary structures unchanged between images . subtraction of the two images leaves an image in which the signal from a volume element ( voxel ) is proportional to the amount of pulmonary arterial blood delivered during the previous heart cycle [ 3 , 511 ] . in principle true fast imaging with steady precession ( true fisp ) sequences in particular has been found to be ideally suited for assessment of pulmonary perfusion . specific types of asl techniques that allow for quantification of regional pulmonary perfusion during a single breath hold are called flow - sensitive alternating inversion recovery ( fair ) and flow - sensitive alternating inversion recovery with an extra radio frequency ( rf ) pulse ( fairer ) . perfusion - weighted images are obtained from the subtraction of a control and tag images , which are interleaved and can be acquired within one breathholding period of 2027 s ( for each slice ) . cardiac motion and pulsatile flow of the descending aorta can generate smearing and flow artefacts , respectively . spatial variation between the control images can lead to ghosting of the blood vessels , and spatial movement between the control and tag images causes the appearance of the adjacent dark - bright pairs of the blood vessels . a closely related technique , called the t1 method , has also been used to measure pulmonary perfusion ( fig . 1 ) [ 14 , 15 ] . it differs primarily in how the experiment is conceptualised as the delivery of a bolus of tagged spins or as an apparent alteration of the longitudinal relaxation time . perfusion - weighted images are acquired in a single shot by completely saturating the static background tissue before image acquisition . the decrease of the lung t1 values in the same slice position from the global to the selective experiment is statistically significant and results from the wash - in effect of the noninverted blood water h spins outside the imaging slice . because the perfusion - weighted image is acquired in a single shot , artefacts arising from patient motion or different respiratory levels can be avoided . furthermore , only very short acquisition times ( 5 s per slice ) are required for the perfusion - weighted image , facilitating rapid and comfortable examinations . this is especially important for lung imaging , as some patients are not able to hold their breath for more than a few seconds.fig . 1arterial spin labeling ( asl ) image in a healthy volunteer ( sagittal orientation , right lung ) . the spatial resolution is 3.9 3.9 10 mm , which allows for visualisation of the major and minor fissure . on the other hand the signal - to - noise ratio is low , making an detailed evaluation difficult arterial spin labeling ( asl ) image in a healthy volunteer ( sagittal orientation , right lung ) . the spatial resolution is 3.9 3.9 10 mm , which allows for visualisation of the major and minor fissure . on the other hand asl - based techniques allow for calculation of relative pulmonary blood flow ( rpbf ) in ml/100 g / min [ 12 , 16 , 17 ] . for conversion of rpbf into pbf the lung density is required , which is usually assumed to be constant with 0.33 g / ml ( at functional residual capacity ) . however normal values are reported to be in the range of 5.5 ml / g / min . using a rapid ir snapshot flash technique , a spin labeling method within the imaging slice was used in combination with a simple two - compartment lung tissue model . the measured pulmonary perfusion rates of the healthy lung parenchyma were on the order of 400 to 600 ml/100 g / min , depending on the subject investigated and the actual slice position in the lung . in healthy volunteers ( n = 6 ) a quantitative comparison between non contrast - enhanced and contrast - enhanced techniques was performed . two coronal slices ( middle and dorsal part of the lung ) were assessed and maps of relative pulmonary blood flow ( rpbf ) were calculated no absolute perfusion measures were calculated . by placing ten regions of interest in each lung , a right - to - left perfusion ratio analysis was performed . for the dorsal slice the middle position however showed a 1643% underestimation of fair - derived rpbf in the right lung . this was explained by tracer saturation for the right lung as the right pulmonary artery is in the plane of both pulses needed for tagging and readout . in an internal control based on the strength and limitations noted above , the clinical application so far has been limited . however , the strength of the techniques lies in the ability to study physiological aspects of the lung perfusion on a regional level [ 2124 ] . important for all perfusion studies is to notice that the snr of asl images is low , particularly in the anterior portions of the lung . therefore , positioning the patient so that the area of interest is down - gravity may increase snr and improve visibility of perfusion defects . also , signal intensity and subsequently perfusion increases during examination on end - expiratory breath - hold compared to end - inspiratory breath - hold [ 25 , 26 ] . normal aging is associated with a decline in pulmonary function and efficiency of gas exchange . the effect of aging on spatial heterogeneity of pulmonary perfusion was studied on 56 healthy , non smoking volunteers with an age range of 2176 years . relative dispersion increased significantly with increasing age by 0.1/decade until age 5059 years , and a significant positive relationship between relative dispersion and age was found . therefore , it was not possible to investigate if the age - related parenchymal changes were the cause for this phenomenon . in an early study keilholz et al . healthy volunteers were placed inside the scanner on their right and left lateral side and coronal images were acquired within a 10-s breath - hold using a fairer haste sequence . in both positions , an increase in the intensity of the dependent lung was found ( 229% for left lateral , 40% for right lateral ) . a pulmonary emboli model was introduced using a balloon catheter that was placed in the left pulmonary artery . after occlusion of the left pulmonary artery a set of fairer images followed by a contrast - enhanced angiography was performed . the huge perfusion defect ( whole left lung ) it has to be noted again , that 8 s were required for a single slice of asl , while the 3d acquisition of the contrast - enhanced perfusion needed 30 s. a similar experiment was done in 5 rabbits were asl perfusion and ventilation images using hyperpolarised 3helium were assessed . after occlusion of the pulmonary artery by a balloon catheter the ventilation was unchanged and the asl perfusion images demonstrated the large perfusion defect . in patients susceptible to high altitude pulmonary oedema ( hape ) it was possible to demonstrate the uneven hypoxic pulmonary vasoconstriction compared to non - susceptible patients by assessing the relative dispersion of blood flow . up to now , only few reports exist on the clinical application of non contrast - enhanced perfusion techniques . due to the lengthy acquisition times and relatively low signal increase , it may be the imaging technique of choice for pregnant women to rule out pulmonary emboli . however , in this stetting other native imaging techniques , like steady - state free precession sequences , also showed a good clinical potential . therefore , the main application will be in exploration of physiological processes where the possibility of unrestricted measurements outweighs the prolonged acquisition time . mri of contrast - enhanced lung perfusion is realised by rapid imaging of the first pass of contrast material through the lungs after intravenous bolus injection . two - dimensional approaches can be acquired in a fast fashion such as 0.3 s for each slice . on the other hand , they lack sufficient spatial resolution and anatomic coverage to characterise most of the perfusion defects or inhomogeneities . for this purpose 3d techniques are mandatory . to visualise the peak enhancement of the lungs , the temporal resolution of contrast - enhanced mri of lung perfusion has to be below the transit time of a contrast bolus through the lungs , being in the range of 3 to 4 s . additionally , for clinical usage a reasonable spatial resolution and anatomic coverage are required to allow for visualisation of perfusion changes on a segmental level . the most frequently used technique is a 3d gradient echo pulse sequence ( flash ) with a short te and tr with a resulting temporal resolution of 1.0 to 1.5 s for each 3d data set , dependent on the used slice thickness of 10 mm down to 5 mm [ 34 , 35 ] . improvements in k - space sampling techniques such as the use of parallel imaging techniques allow for increased spatial resolution without reduction of temporal resolution . a second strategy is to use echo sharing ( like treat ) , where the low frequency k - space data are updated more often than the high frequency k - space data , which is interpolated between consecutive time frames , thus leading to an effective shortening of the total acquisition time . image quality of both techniques was assessed in nine patients . using the same temporal resolution the voxel size was decreased by 24% in the treat data sets compared to the standard perfusion technique ( both techniques used parallel imaging with an acceleration factor of 2 ) . for central and peripheral vascular perception intermodality comparison showed a good agreement ( kappa = 0.74 ) in 14 patients with various diseases ( copd , lung cancer , etc . ) . three k - space acquisition techniques were compared for image quality and snr : ( 1 ) generalised autocalibrating partial parallel acquisition ( grappa ) with an internal acquisition of reference lines , ( 2 ) grappa with a single external acquisition of reference lines before the measurement , and ( 3 ) a combination of grappa with an internal acquisition of reference lines and view sharing . all examinations were performed in ten healthy volunteers ( 5 ml gadubutrol with an injection speed of 2.5 ml / s ) with a constant voxel size : 3.9 3.9 5 mm . at 1.5 t furthermore , the temporal resolution was the shortest with 1.07 s. so far , most of the studies used 0.1 mmol gd - dtpa / kg bodyweight for visualisation of pulmonary perfusion . different amounts of contrast media ( 0.05 mmol / kg bodyweight and 0.1 mmol / kg bodyweight ) , contrast media concentrations ( 0.5 mmol / l and 1 mmol / l ) , and injection protocols ( 2.5 ml / s and 5 ml / s ) were compared in 10 healthy volunteers . by analysing the signal - to - noise ratio no difference was found between the different contrast media concentrations giving the same injection speed . for both contrast agents a dose of 0.1 mmol / kg bodyweight was superior to the half dose protocol . in combination with a rapid injection ( 5 ml / s ) the highest snr in all vascular segments were achieved . regarding the contrast media injection speed , a small bolus profile and fast attenuation of the lung parenchyma is optimal . it was found that with increased speed , the mean transit time ( mtt ) for the pulmonary arteries decreased significantly . however , this decrease was not linear : a four - fold increase in injection rate led to a two - fold decrease in mtt . in the same study it was found , that the cardiac function parameters had no significant influence on the bolus profile . overall , an injection speed of 3 ml / s was recommended for pulmonary perfusion . basic post - processing for improvement of visualisation of perfusion is done by subtraction of the data set with the highest signal intensity from a baseline data set ( fig . 2 ) . in most cases this allows for a rapid analysis of the clinical situation of the patient . however , for any detailed analysis , therapy monitoring or presurgical estimation of the regional pulmonary function , a quantitative analysis is important . a summary of frequently used contrast media injection regimens are provided in table 1.fig . subtraction of both data sets ( c ) results in an image with suppressed background and therefore enhanced perfusion . the 3d nature of the data sets allow for reformation in any plane , like axial ( d ) or sagittal ( e)table 1summary of used contrast media injection protocols and spatial resolution of perfusion - weighted mrinjection speed [ ml / s]dosespatial resolution [ mm]reference2.55 ml gadubutrol3.9 3.9 555 ml magnevist1.9 1.8 1030.05 mmol / kg bw magnevist3.9 3.9 6.340.125 mmol / kg bw magnevist2.9 1.6 1055 ml omniscan1.9 1.8 1250.1 mmol / kg bw magnevist3.5 1.9 450.1 mmol / kg bw magnevist1.9 3.8 3.650.1 mmol / kg bw multihance1.9 3.8 43 - 53 - 5 ml magnevist1.9 1.8 1050.1 mmol / kg bw omniscan1.9 3.6 4 concept of subtraction of perfusion images in a healthy voluneer . subtraction of both data sets ( c ) results in an image with suppressed background and therefore enhanced perfusion . the 3d nature of the data sets allow for reformation in any plane , like axial ( d ) or sagittal ( e ) summary of used contrast media injection protocols and spatial resolution of perfusion - weighted mr quantification of pulmonary perfusion is based on the indicator dilution theory where the maximum of signal intensity and the temporal course of the signal change are used . for quantification an arterial input function ( aif ) has to be defined by placing a region of interest ( roi ) in the main pulmonary artery . the tissue response function of the lung parenchyma can be either determined by multiple single small rois or , better , for the whole lung . assuming a linear relation between the signal and the concentration of contrast agent , signal - time curves are converted to concentration - time curves . hypothesising a negligible amount of extravasating contrast agent during the first pass of the bolus in the lungs , the principles of the indicator dilution theory were applicable . the central volume theorem states the relationship between the perfusion parameters regional pulmonary blood flow ( pbf ml/100 ml lung tissue / min ) , regional pulmonary blood volume ( pbv ml/100 ml lung tissue ) , and the mean transit time ( mtt , s):\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ mtt = \frac{{pbv}}{{pbf } } $ $ \end{document } regional pbv can be calculated by normalising the area under the tissue concentration - time curve to the integral of the arterial input function aif:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ pbv = \frac{{\int { c(t)dt } } } { { \int { aif(t)dt } } } $ $ \end{document } in consideration of the finite length of the bolus and its dispersion during its way to the tissue volume , the relationship between the aif and the tissue concentration - time curve is described by the convolution of the aif and a residue function r(t):\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c(t ) = pbf\int { aif(\tau ) \bullet r(t - \tau ) d\tau } $ $ \end{document}where r(t ) is the amount of contrast agent remaining in the tissue at time t. pbf is therefore the initial height of r(t 0 ) , respectively , the maximum of r(t ) in case of a delay between the aif(t ) and c(t ) , and can be assessed by deconvolving aif(t ) and c(t ) . the deconvolution of the last equation is performed using singular value decomposition ( svd ) , a model - independent nonparametric deconvolution analysis with the potentiality of reducing the noise contribution by applying a suitable threshold to a singular value matrix . this method has shown reliable results for first - pass bolus tracking in clinical studies . however , if the spatial distribution of the underlying lung disease is unknown , or if lung perfusion quantification is required for an entire lung ( e.g. to assess side differences of lung perfusion ) a measurement of a limited lung sample in a small roi will only reflect a poor estimate of lung perfusion . one study used a cross - correlation analysis ( cca ) of perfusion data sets for suppression of central pulmonary vessels . cca has previously been used to improve the snr and arteriovenous separation in time - resolved mr angiography [ 48 , 49 ] . it was shown that it is feasible to identify vessels with a similar signal - intensity time curve as a reference vessel using cca . for this purpose reference rois have to be drawn in the pulmonary artery and pulmonary vein . the quantification of pbv and pbf resulted in an average reduction of 25% for pbv and 15% for pbf compared with the segmentations without vessel exclusion . a comparison of the manual exclusion of vessel structures and the cca revealed an average reduction of 13% for the pbv and 8% for pbf . the reduction by applying the cca was significant ( p 0.001 ) compared with the results with manual and without blood vessel exclusion for all calculated parameters . basically , during quantitative perfusion evaluation the signal - time curves are converted into concentration - time curves . this is especially important for the arterial input function where all contrast media passes by in a compact bolus . a linear relationship , however , is only seen for a small range of concentrations of 3 , 4 or 5 mmol / l as seen in in - vitro experiments [ 5153 ] . the linearity between signal and concentration is dependent on the sequence and parameters used ( like te ) . thus , the use of a saturation recovery prepared flash sequence was proposed , which is often used in myocardial perfusion imaging , to improve the linearity of the measurements and thus the contrast - to - noise ratio ( cnr ) at higher doses . applied to volunteers the usage of 0.057 mmol / kg bodyweight was feasible , while in an animal experiment it was found that already 0.05 mmol gd - dtpa / kg bodyweight resulted in in - vivo concentrations outside the linearity range . however , comparing the quantitative measures of the 0.05 mmol / kg bodyweight the values were too high compared to results from pet where pbf was found to be 121 ml / min/100 ml and pbv 1720 ml/100 ml . in a prospective trial different injection protocols and contrast media concentrations were investigated and compared to quantitative analysis of perfusion spect . the amount of the mr contrast bolus was fixed with 5 ml and an injection speed of 5 ml / s , followed by a 20 ml saline flush . it was found that for patients weighing less than 70 kg a concentration of 0.3 mmol / ml showed the highest accordance to spect and 0.5 mmol / ml for patients weighing more than 70 kg . however , this study used 10-mm - thick slice partitions . therefore , the small amount of 5 ml of contrast media resulted in an appropriate contrast - to - noise ratio . another solution for this complex problem was the usage of a dual bolus approach . in this experiment , the arterial input function is determined by a low volume application of contrast media ( i.e. 0.01 mmol / kg bodyweight ) to achieve a linear relationship between contrast - agent concentration and signal intensity in the large pulmonary vessels . this is followed by boluses with higher doses to obtain sufficient cnr in lung tissue . this may allow absolute quantification as well as the visual analysis of perfusion in lung tissue , which is often not possible when a single low dose is used for absolute perfusion quantification . the drawback of this approach is the small and therefore short bolus ( i.e. 1.5 ml ) for the arterial input function , which requires an extremely high temporal resolution of the perfusion sequence to asses the peak signal . the combination of a small prebolus ( 0.01 mmol / kg bodyweight ) with a main bolus of 0.04 mmol / kg bodyweight resulted in perfusion parameters best comparable with literature data . a combination of the prebolus with a main bolus of 0.08 mmol / kg bodyweight resulted in an underestimation of pbv . for perfusion imaging of the lung , the optimal combination of temporal resolution and contrast - to - noise ratio is essential . it was found that a temporal resolution of approximately 2 s has to be achieved for correct application of the deconvolution algorithm . the influence of noise in the data is especially relevant for the estimates of pbf : high noise levels ( i.e. low cnr values ) lead to a substantial underestimation and a significant loss of accuracy in the pbf estimates . it was concluded that the assessment of pbf might benefit from less aggressive acceleration , for example , by a lower acceleration factor of 2 . the cnr can be notably increased if larger voxel sizes are employed ; the voxel size should thus be chosen as large as clinically acceptable . repeatability of quantitative perfusion measurements was tested in two patient groups and one large volunteer study . in one study ten patients with bronchioalveolar carcinoma were scanned twice within 3 days in inspiratory breath - hold . evaluation was performed by manually placing three regions of interest in the data set . an excellent agreement between both measurements ten patients with pulmonary hypertension were re - scanned 3 weeks after the initial scan . three - dimensional perfusion data sets were acquired in inspiratory breath - hold and evaluation comprised the whole lung . fourteen volunteers were examined twice after 24 h in inspiratory and expiratory breath - hold . no significant difference was found for the pair of measurements performed in expiration and evaluated by the same observer . therefore , for a broad usage of the technique an automatic segmentation approach assisting evaluation should be developed . especially in data sets with pathologically changed perfusion ( i.e. peripheral perfusion defects ) , automatic segmentation algorithms will be difficult to be implemented . these contours were applied to the perfusion data sets after image registration of the haste and perfusion images . this reduced the time needed for evaluation of the perfusion data sets to approximately 10 min . however , this technique was only applied to data sets of healthy volunteers and no reproducibility experiments were performed . so far mr perfusion of the lung parenchyma has been performed at 1.5 t. given the benefits of higher field strengths ( like 3 t ) a higher snr can be expected , which should be beneficial regarding quantification or which allows an increase in spatial resolution . in clinical routine , contrast - enhanced perfusion can be easily applied for visual assessment of disease characteristics . for a qualitative approach the discussion of temporal resolution and contrast media dose is obsolete and it can be performed at any mr scanner . perfusion defects due to vascular obstruction show a typical wedge shape including the lung periphery ( fig . 3 ) . in 41 patients with acute pe the agreement of mr perfusion with single - photon emission computed tomography ( spect ) perfusion for perfusion defects down to the subsegmental level was assessed . mr perfusion showed a very high agreement with spect ( kappa value per examination 0.98 , and 0.98 , 0.83 , and 0.69 for lobar , segmental , and subsegmental perfusion defects , respectively ) . by a quantitative segmental perfusion analysis in patients with acute pulmonary embolism it was possible to determine the obstruction index . this perfusion - based index showed a higher correlation with clinical severity and also was a significant predictor of outcome . this initial report brings mr perfusion in the pole position for being discussed as new modality of choice for pe assessment . furthermore , this radiation - free technique is optimally suited for follow - up studies and treatment monitoring . visual evaluation is also sufficient to differentiate between different causes of pulmonary hypertension ( ph ) like pah and chronic thromboembolic ph ( cteph ) . perfusion was diffusely reduced in pah and focal defects occurred in cteph ( fig . 4 , fig . 5 ) . in 45 patients with moderate to severe copd , perfusion was matched to parenchymal alterations demonstrated by mdct . a high agreement on a lobar level was found between parenchymal destruction and reduction of perfusion in patients with severe emphysema ( kappa of 0.8 ) . in patients with cystic fibrosis the functional score , based on the visual evaluation of the perfusion defects , showed an acceptable intra- and interreader agreement ( concordance correlation coefficient 0.89 and 0.8 , respectively ) .fig . 3coronal 10-mm maximum intensity projection ( mip ) image in a patient in a follow - up examination after acute pulmonary emboli . there is a residual occlusion of the middle lobe leading to a wedge - shaped perfusion defect on the right . there are also residual subtotal obstructions noted in the left upper and lower lobe characterised by a reduced perfusionfig . absolute quantification of perfusion parameters ( a ) pulmonary blood flow and ( b ) pulmonary blood volume . especially the pulmonary blood flow nicely pronounces the area of the perfusion defect as well as the areas with reduced perfusion . original perfusion - weighted data set ( presented as maximum intensity projection ) see fig . 5patient with pulmonary arterial hypertension [ ( a ) pulmonary blood flow and ( b ) pulmonary blood volume ] . however , subpleural perfusion is still present and no wedge - shaped perfusion defects are seen ( c ) original perfusion - weighted data set ( presented as 10 mm maximum intensity projection ) coronal 10-mm maximum intensity projection ( mip ) image in a patient in a follow - up examination after acute pulmonary emboli . there is a residual occlusion of the middle lobe leading to a wedge - shaped perfusion defect on the right . there are also residual subtotal obstructions noted in the left upper and lower lobe characterised by a reduced perfusion same patient as in fig . absolute quantification of perfusion parameters ( a ) pulmonary blood flow and ( b ) pulmonary blood volume . especially the pulmonary blood flow nicely pronounces the area of the perfusion defect as well as the areas with reduced perfusion . original perfusion - weighted data set ( presented as maximum intensity projection ) see fig . 3 patient with pulmonary arterial hypertension [ ( a ) pulmonary blood flow and ( b ) pulmonary blood volume ] . however , subpleural perfusion is still present and no wedge - shaped perfusion defects are seen ( c ) original perfusion - weighted data set ( presented as 10 mm maximum intensity projection ) semi - quantitative analysis of 3d perfusion data sets in eight healthy volunteers allowed for calculation of the left - to - right perfusion ratio ( ratio 0.9 ) , which was confirmed by phase - contrast flow measurements . furthermore , a shorter transit time and higher peak signal were found in the dorsal lung regions . quantitative evaluation of perfusion was compared to perfusion scintigraphy for assessment of left - to - right perfusion ratios in 23 patients with various lung diseases . the mr estimations of left - to - right perfusion ratios correlated significantly with those of perfusion scintigraphy scans ( p < 0.01 ) . the mr ratios computed from pbf showed the highest accuracy , followed by those from peak concentration and pbv . one important clinical field for perfusion assessment is estimation of post - surgical lung function , for example , after lung cancer resection . so far , perfusion scintigraphy is routinely used for this purpose . in 60 patients with lung cancer , mr outperformed scintigraphy for assessment of postoperative fev 1 ( r = 0.93 and r = 0.89 , respectively ) . therefore , it was concluded that perfusion mr is a feasible alternative to pulmonary perfusion scintigraphy for predicting postoperative lung function in patients with lung cancer . however , no ventro - dorsal perfusion gradient was observed , which was explained by the inclusion of central pulmonary vessels in the evaluation . after application of a vasodilatative agent ( inhalation of 100% oxygen ) a significant increase and redistribution of the pulmonary perfusion were found in ten healthy volunteers . therefore , mr perfusion seems to be suited for estimation of therapy response in patients with ph after administration of vasodilatative drugs . in a group of 13 copd patients , the pulmonary perfusion was found to be heterogeneously altered pbf and pbv were significantly reduced and mtt prolonged . despite the pure quantification , another aspect of perfusion , although harder to establish , is the degree of homogeneity . in patients susceptible to high - altitude pulmonary oedema ( hape - s ) the perfusion inhomogeneity was compared between hape resistant volunteers and hape - s patients after 2 h of hypoxia ( being equivalent to an altitude of 4,500 m ) . parameters indicating perfusion inhomogeneity increased during hypoxia in both populations , particularly in hape - s subjects where they increased significantly for almost all evaluations . therefore , this kind of analysis , although not yet broadly used , may be important for future applications in diseases with peripheral vasoconstriction like pulmonary hypertension . so far , only conventional perfusion parameters have been assessed in patients with pulmonary hypertension . compared to healthy , aged - matched volunteers , perfusion parameters are different in patients with pulmonary hypertension , with reduced pbf and pbv and increased mtt [ 74 , 75 ] . only the mtt showed a moderate linear relationship with mpap ( r = 0.54 and r = 0.56 , respectively ) . it remains to be determined if perfusion mr might have a role for follow - up after treatment . however the large data sets are difficult to handle and even visual evaluation might be challenging . therefore , future developments needs to aim at providing easy - to - use post - processing utilities for visual and quantitative evaluation . mri of contrast - enhanced lung perfusion is realised by rapid imaging of the first pass of contrast material through the lungs after intravenous bolus injection . two - dimensional approaches can be acquired in a fast fashion such as 0.3 s for each slice . on the other hand , they lack sufficient spatial resolution and anatomic coverage to characterise most of the perfusion defects or inhomogeneities . for this purpose 3d techniques are mandatory . to visualise the peak enhancement of the lungs , the temporal resolution of contrast - enhanced mri of lung perfusion has to be below the transit time of a contrast bolus through the lungs , being in the range of 3 to 4 s . additionally , for clinical usage a reasonable spatial resolution and anatomic coverage are required to allow for visualisation of perfusion changes on a segmental level . the most frequently used technique is a 3d gradient echo pulse sequence ( flash ) with a short te and tr with a resulting temporal resolution of 1.0 to 1.5 s for each 3d data set , dependent on the used slice thickness of 10 mm down to 5 mm [ 34 , 35 ] . improvements in k - space sampling techniques such as the use of parallel imaging techniques allow for increased spatial resolution without reduction of temporal resolution . a second strategy is to use echo sharing ( like treat ) , where the low frequency k - space data are updated more often than the high frequency k - space data , which is interpolated between consecutive time frames , thus leading to an effective shortening of the total acquisition time . image quality of both techniques was assessed in nine patients . using the same temporal resolution the voxel size was decreased by 24% in the treat data sets compared to the standard perfusion technique ( both techniques used parallel imaging with an acceleration factor of 2 ) . for central and peripheral vascular perception intermodality comparison showed a good agreement ( kappa = 0.74 ) in 14 patients with various diseases ( copd , lung cancer , etc . ) . three k - space acquisition techniques were compared for image quality and snr : ( 1 ) generalised autocalibrating partial parallel acquisition ( grappa ) with an internal acquisition of reference lines , ( 2 ) grappa with a single external acquisition of reference lines before the measurement , and ( 3 ) a combination of grappa with an internal acquisition of reference lines and view sharing . all examinations were performed in ten healthy volunteers ( 5 ml gadubutrol with an injection speed of 2.5 ml / s ) with a constant voxel size : 3.9 3.9 5 mm . at 1.5 t furthermore , the temporal resolution was the shortest with 1.07 s. so far , most of the studies used 0.1 mmol gd - dtpa / kg bodyweight for visualisation of pulmonary perfusion . different amounts of contrast media ( 0.05 mmol / kg bodyweight and 0.1 mmol / kg bodyweight ) , contrast media concentrations ( 0.5 mmol / l and 1 mmol / l ) , and injection protocols ( 2.5 ml / s and 5 ml / s ) were compared in 10 healthy volunteers . by analysing the signal - to - noise ratio no difference was found between the different contrast media concentrations giving the same injection speed . for both contrast agents a dose of 0.1 mmol / kg bodyweight was superior to the half dose protocol . in combination with a rapid injection ( 5 ml / s ) the highest snr in all vascular segments were achieved . regarding the contrast media injection speed , a small bolus profile and fast attenuation of the lung parenchyma is optimal . it was found that with increased speed , the mean transit time ( mtt ) for the pulmonary arteries decreased significantly . however , this decrease was not linear : a four - fold increase in injection rate led to a two - fold decrease in mtt . in the same study it was found , that the cardiac function parameters had no significant influence on the bolus profile . overall , an injection speed of 3 ml / s was recommended for pulmonary perfusion . basic post - processing for improvement of visualisation of perfusion is done by subtraction of the data set with the highest signal intensity from a baseline data set ( fig . 2 ) . in most cases this allows for a rapid analysis of the clinical situation of the patient . however , for any detailed analysis , therapy monitoring or presurgical estimation of the regional pulmonary function , a quantitative analysis is important . a summary of frequently used contrast media injection regimens are provided in table 1.fig . subtraction of both data sets ( c ) results in an image with suppressed background and therefore enhanced perfusion . the 3d nature of the data sets allow for reformation in any plane , like axial ( d ) or sagittal ( e)table 1summary of used contrast media injection protocols and spatial resolution of perfusion - weighted mrinjection speed [ ml / s]dosespatial resolution [ mm]reference2.55 ml gadubutrol3.9 3.9 555 ml magnevist1.9 1.8 1030.05 mmol / kg bw magnevist3.9 3.9 6.340.125 mmol / kg bw magnevist2.9 1.6 1055 ml omniscan1.9 1.8 1250.1 mmol / kg bw magnevist3.5 1.9 450.1 mmol / kg bw magnevist1.9 3.8 3.650.1 mmol / kg bw multihance1.9 3.8 43 - 53 - 5 ml magnevist1.9 1.8 1050.1 mmol / kg bw omniscan1.9 3.6 4 concept of subtraction of perfusion images in a healthy voluneer . subtraction of both data sets ( c ) results in an image with suppressed background and therefore enhanced perfusion . the 3d nature of the data sets allow for reformation in any plane , like axial ( d ) or sagittal ( e ) summary of used contrast media injection protocols and spatial resolution of perfusion - weighted mr quantification of pulmonary perfusion is based on the indicator dilution theory where the maximum of signal intensity and the temporal course of the signal change are used . for quantification an arterial input function ( aif ) has to be defined by placing a region of interest ( roi ) in the main pulmonary artery . the tissue response function of the lung parenchyma can be either determined by multiple single small rois or , better , for the whole lung . assuming a linear relation between the signal and the concentration of contrast agent , signal - time curves are converted to concentration - time curves . hypothesising a negligible amount of extravasating contrast agent during the first pass of the bolus in the lungs , the principles of the indicator dilution theory were applicable . the central volume theorem states the relationship between the perfusion parameters regional pulmonary blood flow ( pbf ml/100 ml lung tissue / min ) , regional pulmonary blood volume ( pbv ml/100 ml lung tissue ) , and the mean transit time ( mtt , s):\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ mtt = \frac{{pbv}}{{pbf } } $ $ \end{document } regional pbv can be calculated by normalising the area under the tissue concentration - time curve to the integral of the arterial input function aif:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ pbv = \frac{{\int { c(t)dt } } } { { \int { aif(t)dt } } } $ $ \end{document } in consideration of the finite length of the bolus and its dispersion during its way to the tissue volume , the relationship between the aif and the tissue concentration - time curve is described by the convolution of the aif and a residue function r(t):\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c(t ) = pbf\int { aif(\tau ) \bullet r(t - \tau ) d\tau } $ $ \end{document}where r(t ) is the amount of contrast agent remaining in the tissue at time t. pbf is therefore the initial height of r(t 0 ) , respectively , the maximum of r(t ) in case of a delay between the aif(t ) and c(t ) , and can be assessed by deconvolving aif(t ) and c(t ) . the deconvolution of the last equation is performed using singular value decomposition ( svd ) , a model - independent nonparametric deconvolution analysis with the potentiality of reducing the noise contribution by applying a suitable threshold to a singular value matrix . this method has shown reliable results for first - pass bolus tracking in clinical studies . however , if the spatial distribution of the underlying lung disease is unknown , or if lung perfusion quantification is required for an entire lung ( e.g. to assess side differences of lung perfusion ) a measurement of a limited lung sample in a small roi will only reflect a poor estimate of lung perfusion . one study used a cross - correlation analysis ( cca ) of perfusion data sets for suppression of central pulmonary vessels . cca has previously been used to improve the snr and arteriovenous separation in time - resolved mr angiography [ 48 , 49 ] . it was shown that it is feasible to identify vessels with a similar signal - intensity time curve as a reference vessel using cca . for this purpose reference rois have to be drawn in the pulmonary artery and pulmonary vein . the quantification of pbv and pbf resulted in an average reduction of 25% for pbv and 15% for pbf compared with the segmentations without vessel exclusion . a comparison of the manual exclusion of vessel structures and the cca revealed an average reduction of 13% for the pbv and 8% for pbf . the reduction by applying the cca was significant ( p 0.001 ) compared with the results with manual and without blood vessel exclusion for all calculated parameters . basically , during quantitative perfusion evaluation the signal - time curves are converted into concentration - time curves . this is especially important for the arterial input function where all contrast media passes by in a compact bolus . a linear relationship , however , is only seen for a small range of concentrations of 3 , 4 or 5 mmol / l as seen in in - vitro experiments [ 5153 ] . the linearity between signal and concentration is dependent on the sequence and parameters used ( like te ) . thus , the use of a saturation recovery prepared flash sequence was proposed , which is often used in myocardial perfusion imaging , to improve the linearity of the measurements and thus the contrast - to - noise ratio ( cnr ) at higher doses . applied to volunteers the usage of 0.057 mmol / kg bodyweight was feasible , while in an animal experiment it was found that already 0.05 mmol gd - dtpa / kg bodyweight resulted in in - vivo concentrations outside the linearity range . however , comparing the quantitative measures of the 0.05 mmol / kg bodyweight the values were too high compared to results from pet where pbf was found to be 121 ml / min/100 ml and pbv 1720 ml/100 ml . in a prospective trial different injection protocols and contrast media concentrations were investigated and compared to quantitative analysis of perfusion spect . the amount of the mr contrast bolus was fixed with 5 ml and an injection speed of 5 ml / s , followed by a 20 ml saline flush . it was found that for patients weighing less than 70 kg a concentration of 0.3 mmol / ml showed the highest accordance to spect and 0.5 mmol / ml for patients weighing more than 70 kg . however , this study used 10-mm - thick slice partitions . therefore , the small amount of 5 ml of contrast media resulted in an appropriate contrast - to - noise ratio . another solution for this complex problem was the usage of a dual bolus approach . in this experiment , the arterial input function is determined by a low volume application of contrast media ( i.e. 0.01 mmol / kg bodyweight ) to achieve a linear relationship between contrast - agent concentration and signal intensity in the large pulmonary vessels . this is followed by boluses with higher doses to obtain sufficient cnr in lung tissue . this may allow absolute quantification as well as the visual analysis of perfusion in lung tissue , which is often not possible when a single low dose is used for absolute perfusion quantification . the drawback of this approach is the small and therefore short bolus ( i.e. 1.5 ml ) for the arterial input function , which requires an extremely high temporal resolution of the perfusion sequence to asses the peak signal . the combination of a small prebolus ( 0.01 mmol / kg bodyweight ) with a main bolus of 0.04 mmol / kg bodyweight resulted in perfusion parameters best comparable with literature data . a combination of the prebolus with a main bolus of 0.08 mmol / kg bodyweight resulted in an underestimation of pbv . for perfusion imaging of the lung , the optimal combination of temporal resolution and contrast - to - noise ratio is essential . it was found that a temporal resolution of approximately 2 s has to be achieved for correct application of the deconvolution algorithm . the influence of noise in the data is especially relevant for the estimates of pbf : high noise levels ( i.e. low cnr values ) lead to a substantial underestimation and a significant loss of accuracy in the pbf estimates . it was concluded that the assessment of pbf might benefit from less aggressive acceleration , for example , by a lower acceleration factor of 2 . the cnr can be notably increased if larger voxel sizes are employed ; the voxel size should thus be chosen as large as clinically acceptable . repeatability of quantitative perfusion measurements was tested in two patient groups and one large volunteer study . in one study ten patients with bronchioalveolar carcinoma were scanned twice within 3 days in inspiratory breath - hold . evaluation was performed by manually placing three regions of interest in the data set . an excellent agreement between both measurements ten patients with pulmonary hypertension were re - scanned 3 weeks after the initial scan . three - dimensional perfusion data sets were acquired in inspiratory breath - hold and evaluation comprised the whole lung . fourteen volunteers were examined twice after 24 h in inspiratory and expiratory breath - hold . no significant difference was found for the pair of measurements performed in expiration and evaluated by the same observer . therefore , for a broad usage of the technique an automatic segmentation approach assisting evaluation should be developed . especially in data sets with pathologically changed perfusion ( i.e. peripheral perfusion defects ) , automatic segmentation algorithms will be difficult to be implemented . these contours were applied to the perfusion data sets after image registration of the haste and perfusion images . this reduced the time needed for evaluation of the perfusion data sets to approximately 10 min . however , this technique was only applied to data sets of healthy volunteers and no reproducibility experiments were performed . so far mr perfusion of the lung parenchyma has been performed at 1.5 t. given the benefits of higher field strengths ( like 3 t ) a higher snr can be expected , which should be beneficial regarding quantification or which allows an increase in spatial resolution . in clinical routine , contrast - enhanced perfusion can be easily applied for visual assessment of disease characteristics . for a qualitative approach the discussion of temporal resolution and contrast media dose is obsolete and it can be performed at any mr scanner . perfusion defects due to vascular obstruction show a typical wedge shape including the lung periphery ( fig . 3 ) . in 41 patients with acute pe the agreement of mr perfusion with single - photon emission computed tomography ( spect ) perfusion for perfusion defects down to the subsegmental level was assessed . mr perfusion showed a very high agreement with spect ( kappa value per examination 0.98 , and 0.98 , 0.83 , and 0.69 for lobar , segmental , and subsegmental perfusion defects , respectively ) . by a quantitative segmental perfusion analysis in patients with acute pulmonary embolism it was possible to determine the obstruction index . this perfusion - based index showed a higher correlation with clinical severity and also was a significant predictor of outcome . this initial report brings mr perfusion in the pole position for being discussed as new modality of choice for pe assessment . furthermore , this radiation - free technique is optimally suited for follow - up studies and treatment monitoring . visual evaluation is also sufficient to differentiate between different causes of pulmonary hypertension ( ph ) like pah and chronic thromboembolic ph ( cteph ) . perfusion was diffusely reduced in pah and focal defects occurred in cteph ( fig . 4 , fig . 5 ) . in 45 patients with moderate to severe copd , perfusion was matched to parenchymal alterations demonstrated by mdct . a high agreement on a lobar level was found between parenchymal destruction and reduction of perfusion in patients with severe emphysema ( kappa of 0.8 ) . in patients with cystic fibrosis the functional score , based on the visual evaluation of the perfusion defects , showed an acceptable intra- and interreader agreement ( concordance correlation coefficient 0.89 and 0.8 , respectively ) .fig . 3coronal 10-mm maximum intensity projection ( mip ) image in a patient in a follow - up examination after acute pulmonary emboli . there is a residual occlusion of the middle lobe leading to a wedge - shaped perfusion defect on the right . there are also residual subtotal obstructions noted in the left upper and lower lobe characterised by a reduced perfusionfig . absolute quantification of perfusion parameters ( a ) pulmonary blood flow and ( b ) pulmonary blood volume . especially the pulmonary blood flow nicely pronounces the area of the perfusion defect as well as the areas with reduced perfusion . original perfusion - weighted data set ( presented as maximum intensity projection ) see fig . 3fig . 5patient with pulmonary arterial hypertension [ ( a ) pulmonary blood flow and ( b ) pulmonary blood volume ] . however , subpleural perfusion is still present and no wedge - shaped perfusion defects are seen ( c ) original perfusion - weighted data set ( presented as 10 mm maximum intensity projection ) coronal 10-mm maximum intensity projection ( mip ) image in a patient in a follow - up examination after acute pulmonary emboli . there is a residual occlusion of the middle lobe leading to a wedge - shaped perfusion defect on the right . there are also residual subtotal obstructions noted in the left upper and lower lobe characterised by a reduced perfusion same patient as in fig . absolute quantification of perfusion parameters ( a ) pulmonary blood flow and ( b ) pulmonary blood volume . especially the pulmonary blood flow nicely pronounces the area of the perfusion defect as well as the areas with reduced perfusion . original perfusion - weighted data set ( presented as maximum intensity projection ) see fig . 3 patient with pulmonary arterial hypertension [ ( a ) pulmonary blood flow and ( b ) pulmonary blood volume ] . however , subpleural perfusion is still present and no wedge - shaped perfusion defects are seen ( c ) original perfusion - weighted data set ( presented as 10 mm maximum intensity projection ) semi - quantitative analysis of 3d perfusion data sets in eight healthy volunteers allowed for calculation of the left - to - right perfusion ratio ( ratio 0.9 ) , which was confirmed by phase - contrast flow measurements . furthermore , a shorter transit time and higher peak signal were found in the dorsal lung regions . quantitative evaluation of perfusion was compared to perfusion scintigraphy for assessment of left - to - right perfusion ratios in 23 patients with various lung diseases . the mr estimations of left - to - right perfusion ratios correlated significantly with those of perfusion scintigraphy scans ( p < 0.01 ) . the mr ratios computed from pbf showed the highest accuracy , followed by those from peak concentration and pbv . one important clinical field for perfusion assessment is estimation of post - surgical lung function , for example , after lung cancer resection . so far , perfusion scintigraphy is routinely used for this purpose . in 60 patients with lung cancer , mr outperformed scintigraphy for assessment of postoperative fev 1 ( r = 0.93 and r = 0.89 , respectively ) . therefore , it was concluded that perfusion mr is a feasible alternative to pulmonary perfusion scintigraphy for predicting postoperative lung function in patients with lung cancer . however , no ventro - dorsal perfusion gradient was observed , which was explained by the inclusion of central pulmonary vessels in the evaluation . after application of a vasodilatative agent ( inhalation of 100% oxygen ) a significant increase and redistribution of the pulmonary perfusion were found in ten healthy volunteers . therefore , mr perfusion seems to be suited for estimation of therapy response in patients with ph after administration of vasodilatative drugs . in a group of 13 copd patients , the pulmonary perfusion was found to be heterogeneously altered pbf and pbv were significantly reduced and mtt prolonged . despite the pure quantification , another aspect of perfusion , although harder to establish , is the degree of homogeneity . in patients susceptible to high - altitude pulmonary oedema ( hape - s ) the perfusion inhomogeneity was compared between hape resistant volunteers and hape - s patients after 2 h of hypoxia ( being equivalent to an altitude of 4,500 m ) . parameters indicating perfusion inhomogeneity increased during hypoxia in both populations , particularly in hape - s subjects where they increased significantly for almost all evaluations . therefore , this kind of analysis , although not yet broadly used , may be important for future applications in diseases with peripheral vasoconstriction like pulmonary hypertension . so far , only conventional perfusion parameters have been assessed in patients with pulmonary hypertension . compared to healthy , aged - matched volunteers , perfusion parameters are different in patients with pulmonary hypertension , with reduced pbf and pbv and increased mtt [ 74 , 75 ] . only the mtt showed a moderate linear relationship with mpap ( r = 0.54 and r = 0.56 , respectively ) . it remains to be determined if perfusion mr might have a role for follow - up after treatment . however the large data sets are difficult to handle and even visual evaluation might be challenging . therefore , future developments needs to aim at providing easy - to - use post - processing utilities for visual and quantitative evaluation . non contrast - enhanced techniques for assessment of pulmonary perfusion are an important tool for understanding of lung physiology . given the low spatial resolution and limited signal - to - noise ratio , a broad clinical application appears to be difficult . contrast - enhanced perfusion techniques are broadly available and are easily and quickly performed , even in critical ill patients .

rituximab is a chimeric monoclonal antibody employed for the treatment of cd20-positive b - cell non - hodgkin 's lymphoma , chronic lymphocytic leukemia , rheumatoid arthritis , granulomatosis with polyangiitis and microscopic polyangiitis . it binds specifically to the cd20 antigen expressed on pre - b and consequently on mature b - lymphocytes of both normal and malignant cells , inhibiting their proliferation through apoptosis , cdc , and adcc mechanisms . the immunomodulatory activity of rituximab is closely related to critical quality attributes that characterize its chemical composition and spatial configuration , which determine the recognition of cd20 and the binding to receptors or factors involved in its effector functions , while regulating the potential immunogenic response . herein , we present a physicochemical and biological characterization followed by a pharmacodynamics and immunogenicity study to demonstrate comparability between two products containing rituximab . the physicochemical and biological characterization revealed that both products fit within the same response intervals exhibiting the same degree of variability . with regard to clinical response , both products depleted cd20 + b - cells until posttreatment recovery and no meaningful differences were found in their pharmacodynamic profiles . the evaluation of anti - chimeric antibodies did not show differential immunogenicity among products . overall , these data confirm that similarity of critical quality attributes results in a comparable immunomodulatory activity .

rituximab is a chimeric monoclonal antibody ( mab ) approved by the fda on 1997 as single agent for the treatment of relapsed or refractory , low - grade or follicular cd20-positive b - cell non - hodgkin 's lymphoma ( nhl ) and later , in 2006 , as a treatment in combination with cyclophosphamide , doxorubicin , vincristine , and prednisone ( chop ) or other anthracycline - based chemotherapy regimens for patients with diffuse large b - cell lymphoma ( dlbcl ) . in both cases it increases the response rate , diminishes disease progression events , and augments patients survival [ 13 ] . the molecular weight of rituximab is 144,544 da and is constituted of 1328 aa . as an igg isotype 1/kappa , rituximab contains a conserved n - glycosylation site at asn297 of both heavy chains and is occupied by biantennary glycan structures , while murine variable regions and human constant regions define its chimeric nature . rituximab mechanisms of action comprise the binding of its fab domain to cd20 + b - lymphocytes for the induction of apoptosis , either directly or throughout the recruitment of immune effector functions by its fc domain , thus mediating b - cell lysis through complement - dependent cytotoxicity mechanism ( cdc ) , after binding to c1q , or antibody - dependent cellular cytotoxicity mechanism ( adcc ) once is recognized by the fc receptors ( fcrs ) of effector cells , including natural killers , granulocytes , and macrophages [ 46 ] . besides , the current knowledge concerning monoclonal antibodies ( mab ) permits us to correlate the immunomodulatory activity of a mab to critical quality attributes ( cqas ) that depict its chemical composition and spatial configuration . on this regard , rituximab cqas are associated with the appropriate recognition of cd20 + b - cells and the achievement of effector functions . nevertheless , rituximab is subject to posttranslational modifications that can be acquired during its lifecycle , which provides an inherent physicochemical heterogeneity that could impact on its functionality [ 7 , 8 ] . although this heterogeneity is expected to occur batch to batch , its variability breadth can be controlled during the manufacturing process ; thus , an acceptance range should be established for each cqa , depending on the observed safety and efficacy for the given process capabilities . this is particularly important for the development of follow - on products , for which the demonstration of highly similar cqas variability , along with the demonstration of comparable pharmacological responses with respect to the reference product , grant the biosimilar denomination [ 1012 ] . it is reported that acidic and basic isoforms , coming mainly from oxidation , deamidation , isomerization , amination , cyclization , glycation , and the presence of c - terminal lysines , could alter the mab affinity to target and receptor molecules due to the modification of electrostatic and hydrophobic interactions with cell membranes . on the other hand , glycosylation contributes in maintaining stability of the mabs ' three - dimensional structure and modulates the binding interaction of the fc domain to the effector cells , influencing cdc and adcc mechanisms . regarding its immunogenicity , rituximab is considered as a low risk molecule although potentially immunogenic , since it does not exhibit cross - reactions with endogenous antibodies or autoimmunity induction ; however , due to its chimeric nature , the production of human anti - chimeric antibodies ( hacas ) may lead to the loss of efficacy in certain cases . consequently , to discard any differential immunogenic response of a biosimilar rituximab , the comparability of its chemical composition ( i.e. , sequence and posttranslational modifications ) should be demonstrated [ 15 , 16 ] . aggregation is another attribute that has been also identified as a cqa that participates in the development of an immunogenic response . in this work , we conducted a comprehensive characterization followed up by a pharmacodynamics - immunogenicity clinical study of two products containing rituximab . the characterization exercise is focused on the comparison between the cqas associated with the pharmacodynamic profile ( pd ) and the potential immunogenicity of rituximab such as protein identity ( amino acid sequence ) , charge and glycosylation heterogeneity , aggregates content , and binding affinity to fcriia and fcriiia , while the biological characterization included measurement of the affinity to cd20 and potency through adcc and cdc . the clinical evaluation was intended to demonstrate that both products exhibit the same behaviour as the result of a high physicochemical comparability . dibasic sodium phosphate heptahydrate ( na2hpo47h2o ) , monobasic sodium phosphate monohydrate ( nah2po4h2o ) , sodium chloride ( nacl ) , tris - hydrochloride ( nh2c(c2oh)3hcl ) , and sodium hydroxide ( naoh ) were obtained from j. t. baker ( center valley , pa ) . sodium azide ( nan3 ) , ammonium formate ( ch5no2 ) , rpmi-1640 medium , fetal bovine serum ( fbs ) , and formic acid were acquired from sigma - aldrich ( st . louis , mo ) . ( hayward , ca ) ; pngase f was purchased from new england biolabs ( woburn , ma ) and human igg - fc antibody from bethyl laboratories inc . tetramethylbenzidine ( tmb ) substrate was obtained from thermo scientific ( waltham , ma ) . adcc reporter bioassay kit and celltiter 96 mtt were purchased from promega ( madison , wi ) . water was obtained from a millipore milli - q biocel system ( billerica , ma ) . two products containing rituximab were employed : kikuzubam from probiomed s.a . de c.v . , ; manchester , uk ) coupled to an acquity uplc h - class bio system ( waters corp . , chromatographic separation was carried out using an acquity uplc h - class bio system with a linear gradient from 22 to 50% of acetonitrile using 100 mm ammonium formate aqueous solution at ph 4.50 as mobile phase a. fluorescence detection was set at an excitation wavelength of 250 nm and 420 nm for emission , using a 150 2.1 mm , 1.7 m acquity uplc beh glycan column coupled with a 1.7 m vanguard beh glycan precolumn from waters corp . rituximab purity was assessed on a 4.6 mm 300 mm acquity ethylene bridged hybrid 200 analytical column with particle and pore diameters of 1.7 m and 200 , respectively ( waters corp . , milford , ma ) . 20 mm phosphate buffer containing 150 mm nacl and 3 mm nan3 at ph 6.8 was used as mobile phase with isocratic gradient . uv detector was set at 280 nm in an acquity uplc h - class bio system . affinity constants under equilibrium ( ka ) were obtained by isothermal titration calorimetry ( itc ) using a nano itc instrument from ta instruments inc . ( new castle , de ) . 300 l of fcriia and fcriiia solutions at 5.0 m in pbs at ph 7.0 was titrated with continuous injections of 1.9 l rituximab solutions at 50 m in pbs at ph 7.0 until saturation at 25c . ; new castle , de ) was used for the integration of heat signals and nonlinear regression analysis of the data . wil2-s cell line ( atcc : crl-8885 ) that expresses the cd20 antigen was incubated in the presence of different concentrations of rituximab in rpmi-1640 medium with 10% fbs for 2 h at 37c . a secondary antibody ( anti - human igg - fc ) coupled to a radish peroxidase was added to detect the rituximab - wil2-s complex after 1 h of incubation at 37c , using tmb as substrate for 30 min at room temperature . the test results were expressed as the relative percentage of the ec50 from the concentration - response curve of kikuzubam with respect to the reference product . cd20 positive cells ( wil2-s , atcc crl-8885 ) were incubated in rpmi 1640 media with 10% of fbs with different concentrations of rituximab and complement human serum for 4 h at 37c and 5% co2 . then mts substrate was added to each well with a further incubation of 2 h at the same conditions . the result of the assay was expressed as % relative potency , which is obtained comparing to the ec50 of the dose - response curve of kikuzubam with respect of the ec50 of the dose - response curve of the reference product . the adcc reporter bioassay kit from promega ( madison , wi ) positive cells ( wil2-s , atcc crl-8885 ) were incubated with different concentrations of test antibody and a specific concentration of jurkat transformed cells expressing cd16 . the result of the assay was expressed as % of relative potency of kikuzubam with respect to the reference product . a double - blind two arms ( 1 and 2 ) were crossed after three cycles of treatment in order to review the expected use conditions of kikuzubam and the possible impact on its efficacy as suggested by the mexican health authorities . the study protocol was approved by the irb / iec ( institutional review board / independent committee ) of the participating research centres and by the mexican health authorities ( study protocol codes cas / or/01/cmn/0833004101444 - 0114/2009 and cas / or/01/cmn/07330021830339 - 0816/2008 ) . the study was conducted in accordance with the regulations and ethical principles based on the declaration of helsinki , the principles of the international conference on harmonization ( ich ) , and the guidelines for good clinical practice ( gcp ) . an informed consent was obtained from all patients prior to their participation in the study . the aim of the study was to evaluate the biological effects and safety of kikuzubam compared to the reference product during six treatment cycles with chop therapy . patients received either kikuzubam or the reference product in each cycle , according to their treatment group , at a dose of 375 mg / m every 14 days by iv infusion . 59 patients diagnosed with moderate to high degree diffuse cd20 + b - cell non - hodgkin lymphoma were randomly assigned into three groups . group 1 was treated with kikuzubam during the first three cycles and subsequently with the reference product for the remaining three cycles . group 2 was initially treated with the reference product for three cycles and then with kikuzubam for the next three cycles . blood samples were collected from all patients for the determination of cd20 + b - cells levels as the pd endpoint on visits 1 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , and 12 using a cd20 becton dickinson fitc labelling kit in a epic xl beckman coulter inc . additionally , levels of serum human anti - chimeric antibodies ( hacas ) were determined using the human antirituximab ( hada / haca / hama / haha ) igg elisa kit for human from alpha diagnostics ( san antonio , tx ) . the assay precision was determined from the graphs obtained with serum samples , resulting in a coefficient of variation ( cv ) lower than 10% with accuracy ranging from 90 to 110% . analysis of covariance was performed to evaluate the effect of both treatments ( kikuzubam and the reference product ) on the number of cd20 + b - cells relative to basal values ( covariable ) . the anova test was evaluated with a significance level of 0.05 . to avoid the effect of crossing treatments , the cd20 + b - cells depletion analyses were performed considering only the results from the first three cycles of treatment with either kikuzubam or the reference product , in order to compare the response between treatments in a parallel design . the physicochemical properties of rituximab are discussed according to its impact on pd and immunogenicity potential . identity , heterogeneity , purity , and biological activity cqas were studied by comparing several batches of kikuzubam and the reference product . the identity of both products was verified by its tryptic peptide chromatographic profiles followed by ms / ms analyses matched with the theoretical sequence of rituximab ( figure 1 ) . the theoretical sequence was obtained by reverse engineering , comprising a de novo protein sequencing of the reference product by esi - ms / ms and maldi psd using trypsin , glu - c , or asn - n digestions along with edman 's degradation of selected fragments . this sequence was employed for the design and construction of the expression system of kikuzubam ( data not shown ) that revealed inconsistencies in the invention patents [ 21 , 22 ] of rituximab at the amino acid positions 14 and 219 of the heavy chain . our results agree with the sequence published by other groups [ 23 , 24 ] and the united states pharmacopeia . for both products sequence verification , expressed as ms / ms sequence coverage , exceeded the accepted consensus value of 90% , being 98.7% and 98.6% for kikuzubam and 98.7% and 97.2% for the reference product of their heavy and light chains , respectively ( figures 2 and 3 ) . in order to confirm the identity of kikuzubam , exact mass of the whole deglycosylated molecule , coming uniquely from the amino acid sequence , was determined ( table 1 ) . on the other hand , as we previously reported , correspondence between each glycoform and the theoretical mass ( 99.98% ) was observed within and among kikuzubam and the reference product . these results confirm that the primary sequences of both products are identical and also reveal that charge and glycosylation heterogeneities are comparable ; thus , the risk of a differential immunomodulatory response is diminished . the glycosylation heterogeneity of kikuzubam and the reference product was also evaluated as a relevant cqa on the immunomodulatory activity of rituximab . table 2 shows the content of highly mannosylated , hybrid , sialylated , afucosylated and galactosylated glycoforms of both products . it is reported that these glycan isoforms could affect the affinity to the receptors involved in the effector function and stability of a mab , due to charge and steric hindrances . for instance , hybrid ( bisected ) and afucosylated glycans tend to increase the affinity to fc gamma riiia , resulting in an enhanced adcc response [ 27 , 28 ] , while sialylated isoforms could increase immune responses . nonetheless , the glycan heterogeneity of a biosimilar must correspond to the reference product . in this analysis , both products revealed similar glycan heterogeneity , which is consistent with the presence of the same glycoforms observed by the ms analyses of the whole molecule . although minor differences were found in the nonfucosylated and hybrid glycan content between products , no impact was observed on the potency or the efficacy of the adcc assay afterwards ( figure 5 ) [ 14 , 27 , 28 ] . regarding charge heterogeneity , changes higher than 1.0 units in the isoelectric point ( pi ) of a mab could affect its therapeutic activity [ 13 , 29 ] , with the common pi variation observed during manufacturing being from 0.1 to 0.2 pi units the common pi variation observed during manufacturing . on table 3 , we show the pi range , main isoform , and overall calculated pi values of kikuzubam and the reference product . the observed differences were lower than 0.1 pi units confirming comparability of charge heterogeneity among the products . another relevant cqa related to the immunomodulatory activity of rituximab is the aggregation level , which involves the irreversible interaction of two or more denatured protein molecules revealing new epitopes that could stimulate the immune system . a positive correlation between protein aggregation and immunogenicity has been reported for therapeutic proteins , as well as affectations on the biological activity , either directly or indirectly through the formation of neutralizing or binding antibodies . thus , the evaluation of aggregates is an important component of the analytical comparability assessment of therapeutic proteins . the aggregates content of kikuzubam was comparable to the reference product ( table 4 ) , which in both cases complied with the pharmacopeial established limit . in addition to the physicochemical analyses , an extensive biological characterization to assess comparability of the functions ( mechanisms of action ) described for the reference product and kikuzubam was performed through in vitro assays . these studies were designed taking into account the interactions of the fab and fc domains and their associated biological activities described in the literature ( affinity to cd20 , fcriia , and fcriiia ) . the main mechanism of action of rituximab is binding to cd20 [ 4 , 6 ] whose interaction affinity is related to the structure of complementary domain regions ( cdrs ) of the fab fragment , and this reveals the presence of the appropriate chemical and structural properties of this fragment . our results showed that kikuzubam and the reference product have comparable affinities to cd20 ( figure 4 ) . rituximab also can induce the death of cd20 + b - cells by activating effector cells such as natural killer cells ( nk ) , monocytes , and macrophages through the binding of the fcr receptors to its fc domain . clinical studies have shown that the affinity to fcriia and fcriiia receptors is associated with a better response to rituximab in patients with follicular lymphoma . itc results of affinity to fcriia and fcriiia of kikuzubam and the reference product were within the same order of magnitude ( figure 4 ) ; thus , the modulatory functions that lead to b - cell depletion in both products are assumed to follow the same molecular basis . the described fc and fab affinities further modulate cdc and adcc mechanisms of rituximab [ 5 , 6 ] and both were evaluated comparatively for kikuzubam against the reference product ( figure 5 ) . these analyses also confirmed that the physicochemical characteristics of the fc domain of kikuzubam are capable of achieving the same biological functions with comparable potency as the reference product . an abbreviated study conducted on cd20 + non - hodgkin 's lymphoma patients was designed to confirm that the physicochemical and functional characteristics of kikuzubam are adequate to exhibit the same pd profile as the reference product . cd20 was used as the main endpoint . during the treatment , cd20 b - cells were depleted to serum levels lower than 20 cell / ml in the three arms of the study ( figure 6 ) . this is explained by the effect of both rituximab products since the levels of other blood components were recovered within 7 days after the completion of concomitant chop chemotherapy regimen and the application of granulocyte colony stimulating factor ( filgrastim , g - csf ) . cd20 was the only component with no recovery in serum , despite the stimulation after the completion of chemotherapy . once the six cycles of rituximab - chop were completed , a recovery in the serum levels of cd20 + b - cells for the three groups of the study was observed . nonmalignant recovery was demonstrated by pet images as the absence of neoplasms ( data not shown ) . in order to determine the comparability of the primary endpoint , statistical analyses for the three arms before shapiro - wilk test revealed departures from normality of the data ( p < 0.05 ) . mean comparison among the groups was performed by student 's t - test and wilcoxon tests , revealing no significant differences in cd20 + depletion between kikuzubam and the reference product ( p > 0.05 ) ( table 5 ) . combined groups were also analysed using data from arms 1 and 3 to compare all patients treated with kikuzubam against the reference product , one outlier was excluded . the results obtained from this exercise also revealed no meaningful differences among treatments ( figure 6 ) . the production of antichimeric human antibodies ( hacas ) as a result of the loss of tolerance to rituximab by the immune system was evaluated on the three study arms . on arm 1 , two patients showed positive results for the screening test of hacas right after the shift from kikuzubam to the reference product on visit 5 ( table 6 ) . thus , the immunogenic response had to be triggered before the medication shifting , since the first humoral immunogenic response is the production of igm antibodies , which half - life in plasma is approximately four weeks , followed by isotype switching to igg , if loss of tolerance with the consequent hacas production is presented . also , on arm 2 , the presence of hacas in two patients was detected before the shift of treatment ( table 6 ) . then the immunogenic response produced in 4 out of 28 patients from the first two groups can not be considered as a consequence of the drug shifting . the immunogenic response was analogous between kikuzubam and reference product ; therefore , no differential immunogenicity was observed . likewise , three patients from arm 3 presented positive results for the screening hacas test ( table 6 ) . these data suggest that the proportion of patients positive to hacas was comparable between all study arms . the presence of these antibodies did not represent a risk to the patient safety and did not justify abandoning the study . the biological effect was comparable to hacas - negative patients between both products . the hematologic recovery after r - chop cycles , even in patients positive to hacas screening test for both kikuzubam and the reference product , was accomplished within the expected period reported in studies with chemotherapy ; thus , it can be inferred that neither the hacas developed by kikuzubam nor the reference product had a negative effect on the hematologic recovery of patients included in the study . the comprehensive physicochemical , biological , and in vitro characterization studies , including the verification of amino acid sequence , glycosylation and charge heterogeneity , aggregates content , and affinity to cd20 , fciia , and fciiia receptors , provided valuable information to demonstrate comparability between kikuzubam and the reference product . the information provided by these analyses supported the design of a rational clinical evaluation to demonstrate similar immunomodulatory response through the pharmacodynamics and immunogenicity profiles . physicochemical along with biological comparability resulted in a similar immunomodulatory activity between the evaluated products .

migraine is among the 10 most disabling disorders worldwide . it is characterized by episodes of moderate or severe headaches with various degree of disability , resulting in a considerable health burden upon the sufferers and their family . the objective of this article is to review the use of prophylaxis with antiepileptic drugs . particular focus is given to their mechanism of action , metabolism , pharmacokinetics , safety profile , efficacy and to provide a summary of the most relevant clinical studies and patient preference .

migraine is a chronic neurological disorder affecting about 12% of the population with an estimated prevalence of 18.2% in women and 6.5% in men.1,2 its etiology also seems to have a hereditary component.3 over 70% of migraine sufferers have a family history of migraine.4 migraine varies with age and tends to occur most commonly in the second and third decades of life.5 this common and occasionally severe disabling disorder is usually characterized by recurrent headaches frequently unilateral , lasting between 4 and 72 hours , typically aggravated by routine physical activity and often accompanied by a variety of gastrointestinal , neurologic and autonomic symptoms including loss of appetite , nausea , vomiting , photophobia , phonophobia and osmophobia.5,6 approximately one third of migrainous patients experience migraine attacks with aura , typically with visual symptoms ( scintillating shapes , hallucinations , black spots ) preceding or occurring with the attack , but other sensory or language disturbances may occur.7 these transient focal neurological symptoms develop gradually over a period of at least 5 minutes and last less than 1 hour.6 migraine attacks are heterogeneous in regard to the symptomatology , frequency , severity , duration , disability and impact on quality of life , both between different patients and between separate attacks in the individual sufferer.8 from the clinical and economic perspectives , there is a great demand for rapidly acting , effective , safe and abortive headache agents . migraine attacks are believed to be caused by activation of the trigeminal nerve and trigeminovascular system , leading to the release of several neurotransmitters ( calcitonin gene - related peptide , substance p ) that affect vasomotor tone , causing neurogenic inflammation of intracranial and extracranial cerebral vessels.9 the aura is thought to be caused by cortical spreading depression , a slowly propagating wave of intense neuronal and glial depolarization progressing over the cortex and followed by a period of inactivity.10 the most common external triggers for migraine attacks are , in decreasing frequency , stress ( 80% ) , hormonal fluctuations in women ( 65% ) , skipping meals ( 57% ) , changes in weather ( 53% ) , lack of sleep ( 50% ) , perfumes or odors ( 44% ) , neck pain ( 38% ) , certain foods ( 27% ) and physical activity ( 22%).11 in addition to environmental triggers , several genetic factors contribute to migraine pathophysiology.12 family and twin studies have clearly demonstrated that both the rare and common forms of migraine have significant genetic basis . however , approaches to understand this genetic basis have had varying degrees of success.13 recent genetic findings have revealed ion channel and transporter mutations as causative of migraine.7 considerable insights into the pathogenesis of migraine have come from the investigation of the rare autosomal dominant subtype of migraine with aura , familial hemiplegic migraine . three susceptibility genes ( cacna1a , atp1a2 and scna1 ) , which encode either ion channels or ion transport proteins involved in regulating membrane potential , have so far been identified . it is likely that mutations in these genes reduced the threshold for cortical spreading depressions . however , these mutations are not found in typical migraine with aura , suggesting that other ion channels may be involved.7 recently a mutation in the ksnk18 gene , encoding the two - pore domain potassium channel , tresk ( twik - related spinal cord potassium channel ) , has been described in a large family with migraine with aura . tresk modulates neuronal excitability , seems to be involved in pain pathways and is activated by volatile anesthetics , which have been shown to inhibit cortical spreading depression , a key mechanism in the generation of migraine aura . moreover this channel , abundantly expressed in the trigeminal ganglia , controls the sensitivity of pain nerves . this is a possible new approach to the treatment of migraine.4 a wide range of medications have been used for the treatment of acute migraine headache . dihydroergotamine has been used for several decades and produces good relief in 70%80% of patients within 2 h of administration.14 triptans produce similar efficacy.15 despite their widespread use and availability , a significant proportion of patients with migraine are refractory to these agents and require opiate and analgesics for control of acute headache.16 moreover , the presence of cardiovascular risk factors prohibits the use of triptans or ergotamines in many patients.17 recent studies suggest as well the risk of development of chronic daily headaches with the overuse of acute medication including triptans , ergots and other analgesics.18 the management of migraine can be divided into acute ( abortive ) and preventive treatment . patients with frequent and severe headaches often require both approaches.6 preventive therapy is important in reducing migraine morbidity . it should be considered in patients with attacks that are long , severe , frequent ( two or more a month ) , or when there is a marked impact on daily functioning . in addition it may be used for those with adverse events to , or overuse of acute care medicines . the main goal of prophylaxis is the reduction in frequency , intensity and duration of migraine attacks as well as the prevention of chronicity . however , good prophylaxis will usually result in better response to acute treatment , with improved daily function and reduction in disability . the ultimate target is to improve the quality of life and patient s performance in daily activities and work productivity , and to reduce healthcare costs . when choosing a prophylactic treatment , one should take into account its efficacy , tolerability and the existence of comorbid conditions.6,1921 currently , the recommended first - line agents come from different pharmacological classes with primary indications that are usually approved for other medical conditions . they include the beta - adrenoceptor blocking drugs propranolol and metoprolol , the antidepressant amitriptyline , the calcium channel blocker flunarizine and most recently the neuromodulator drugs valproate and topiramate that are playing an increasingly important role . second line drugs have either been less effective in clinical trials or have only been tested in a small number of less well - designed trials and require further investigation . table 1 lists preferred prophylactic agents according to their efficacy , strength of evidence and frequency of side effects.22,23 it is proposed that epilepsy and migraine share some of the same pathophysiological mechanisms ( table 2 ) including abnormal function of voltage - gated sodium and calcium channels and an imbalance between gaba - mediated inhibition and excitatory glutamate - mediated transmission.24 evidence suggests that epilepsy is a comorbid condition of migraine . it occurs more commonly in patients with migraine than in the general population , and the prevalence of migraine in epileptic patients is higher than in controls.25 the use of antiepileptic drugs for migraine prevention is well known and the effectiveness of these medications has been demonstrated in several clinical trials . however , only a few anti - epileptic drugs have been shown to be effective in migraine prophylaxis . valproate and topiramate are effective and well tolerated in migraine prevention and are suitable first - line agents . on the other hand , acetazolamide , lamotrigine , oxcarbazepine and vigabatrin are not effective . the efficacy of gabapentin in migraine prevention is variable ; it is not considered to be a first line treatment and requires further evaluation.8,23 the antiepileptic drugs , also referred to as neuro - modulators , appear to act in migraine by targeting multiple molecular sites in the brain ( figs . 1 and 2 ) , altering neurotransmission through their effects on ion channels , neurotransmitter receptors , and neurotransmitter metabolism.3,26 the interaction with these multiple sites decreases abnormal brain excitability and protects vulnerable neurons in conditions with a high - energy demand , such as neuronal hyper - activity as well as metabolic impairment.27 valproate is available as valproic acid , divalproex sodium or both formulations , without differences in efficacy between the various forms . 3a ) stops or prevents migraine is not clearly understood.2 valproate has been shown to have both central and peripheral mechanisms of action that may have relevance in the migraine cascade.28 ( 1 ) it increases gamma - aminobutyric acid ( gaba ) levels in synaptosomes and in the brain , via activation of glutamic acid decarboxylase ( gaba - synthetic enzyme ) and via inhibition of gaba aminotransferase and succinate semialdehyde dehydrogenase ( gaba - degradative enzymes).29 ( 2 ) it inhibits the voltage - sensitive calcium channels ( t - type).2 ( 3 ) it interacts with central 5-ht system thereby reducing the effect of inflammation of serotoninergic neurons in the brainstem . ( 4 ) it reduces the central trigeminal nerve activation ( by increasing gaba levels).25 ( 5 ) finally , valproate was shown to reduce experimental neurogenic inflammation in the peripheral trigeminal vascular system , an effect that is mediated through gabaa receptor agonism.28 valproate is effective for long - term migraine prophylaxis , and initial benefits are maintained for periods in excess of 3 years.30 it is rapidly absorbed in the gastrointestinal tract and metabolized almost entirely by the liver . the relationship between dose and plasma concentration is not linear and its bioavailability depends on the formulation and dosing regimen.31 topiramate is a sulfamate - substituted monosaccharide derived from the naturally occurring sugar d - fructose ( fig . its antimigraine potential is based on several possible mechanisms of action namely : ( 1 ) modifies the excitability of nerves by blocking voltage - sensitive sodium channels and l - type voltage - activated calcium channels.27,28 ( 2 ) inhibits carbonic anhydrase activity.32 ( 3 ) inhibits the excitatory glutamate pathway while enhancing the inhibitory effect of gaba.30 the actual mechanism of action for its antimigraine activity is not known.5 it is rapidly and almost completely absorbed from the gastrointestinal tract with tmax between 1 and 4 hours , has a high oral bioavailability ( 81% to 95% ) while being virtually unaffected by food . it has a low level of binding to plasma proteins ( about 10%20% ) and does not significantly inhibit or induce other drug metabolizing enzymes . it is not extensively metabolized ( approximately 20% is metabolized in the liver ) and readily enters the central nervous system ( cns ) . it shows linear steady - state pharmacokinetics and has a long half - life ranging from 19 to 25 hours.3133 zonisamide is a sulphonamide derivative ( fig . this new generation of antiepileptic drugs has been available in japan for over 10 years and has only recently been introduced into the usa and europe . it has some mechanisms of action very similar to those of topiramate including blockade of voltage - gated sodium channels , inhibition of carbonic anhydrase , inhibition of potassium - mediated release of glutamate , and enhancement of gaba release . furthermore , it has a specific mechanism of action by reducing ion flow through t - type calcium channels.19,33,34 it also reduces nitric oxide production and scavenges nitric oxide free radicals.1 it is well tolerated and presents a favorable pharmacokinetic profile for clinical use . it is rapidly and completely absorbed from the gastrointestinal tract , with peak plasma concentrations occurring 2 to 4 hours following its administration . it has a long plasma elimination half - life ( 63 to 69 hours in healthy volunteers ) allows reduced frequency of administration . since it is metabolized by the cytochrome p450 3a4 , other drugs inducing or inhibiting this enzyme may affect its plasma elimination.35,36 gabapentin ( fig . it binds with high affinity to two of the four known 2 subunits ( 21 and 22 ) of voltage - gated calcium channels , producing an inhibition of high - voltage - activated calcium currents and resulting in reduction of synaptic transmission.37 it has a very attractive pharmacokinetic profile . it is not bound to plasma proteins , does not have significant drug - drug interactions , does not induce hepatic enzymes and is not metabolized . the effectiveness of valproate in migraine prophylaxis was first reported in an open - label study,38 with another open - label study demonstrating decreased severity and frequency of headaches in migrainous patients.39 its efficacy has been demonstrated in three randomized double - blind controlled studies.4244 in the first trial , 107 patients were treated with valproate ( n = 70 ) or placebo ( n = 37 ) respectively , during a period of 3 months . valproate was started at a dose of 250 mg / day and then titrated gradually to achieve a plasma concentration of approximately 70 to 120 mg / l . the number of participants with a 50% or greater reduction in headache frequency with valproate was significantly better ( 48% ) than with placebo ( 14%).40 the efficacy and safety of valproate was evaluated in the second study during a 3 month period . a total of 176 patients were randomized into four groups : placebo ( n = 44 ) , valproate 500 mg / day ( n = 45 ) , 1000 mg / day ( n = 43 ) and 1500 mg / day ( n = 44 ) . the initial dose for valproate - treated patients was 250 mg / day and was then increased by 250 mg every 4 days ( every 8 days for the 500 mg group ) until the assigned randomized dose was achieved . the monthly migraine frequency decreased significantly for the 500-mg / day group ( from 4.5 to 2.8 ) , the 1000-mg / day group ( from 4.7 to 2.7 ) and the 1500-mg / day group ( from 4.7 to 3.0 ) versus the placebo group ( from 6.1 to 5.6 ) . however the overall median reduction of 38% in the valproate 1000-mg / day group was not significantly greater than the overall 19% median reduction observed in the placebo group.41 in the third study 234 patients were treated during a period of 17 weeks with valproate ( n = 119 ) or matching placebo ( n = 115 ) . the treatment was initiated at 500 mg once daily for 1 week , and the dose was then increased to 1000 mg during the second week . mean migraine frequency decreased significantly for the valproate treated group ( from 5.8 to 3.7 ) versus the placebo group ( from 6.3 to 4.6).42 the efficacy of topiramate in migraine prevention was initially shown in small preliminary studies,43,44 and thereafter established in three pivotal multi - centre , randomized , double - blind , placebo - controlled trials.45,47,48 the first was a multicentre 6-month trial performed in 49 locations in the usa . a total of 487 patients were randomized to four groups : placebo ( n = 117 ) , topiramate 50 mg / day ( n = 125 ) , 100 mg / day ( n = 128 ) or 200 mg / day ( n = 117 ) . topiramate was started at 25 mg / day and increased by 25 mg / week for 8 weeks until the maximum assigned dose given in divided doses in the morning and evening was reached . the mean monthly migraine frequency decreased significantly for the 100-mg / day group ( from 6.4 2.7 to 3.7 3.3 ; p < 0.001 ) and the 200-mg / day group ( from 6.6 3.1 to 3.9 3.4 ; p < 0.001 ) versus the placebo group ( from 6.4 2.6 to 5.3 3.6 ) , but not for the 50-mg / day group ( from 5.8 2.5 to 4.5 3.1 ; p = 0.12 ) . topiramate - treated patients ( 50 mg / day : 35.9% ( p = 0.04 ) ; 100 mg / day : 54% ( p < 0.001 ) ; 200 mg / day : 52.3% ( p < 0.001 ) ) exhibited a 50% or more reduction in monthly migraine frequency ( responder rate ) than placebo - treated patients ( 22.6%).45 the second trial was conducted during a period of 26 weeks at 52 north american clinical centers . a total of 483 patients were randomized into four groups : placebo ( n = 114 ) , topiramate 50 mg / day ( n = 116 ) , 100 mg / day ( n = 120 ) or 200 mg / day ( n = 117 ) . mean monthly migraine frequency decreased significantly for patients receiving topiramate at 100 mg / day ( from 5.8 2.6 to 3.5 3.5 ; p = 0.008 ) and at 200 mg / day ( from 5.1 2.0 to 3.0 2.2 ; p < 0.001 ) versus the placebo group ( from 5.6 2.2 to 4.5 2.9 ) . topiramate - treated patients ( 50 mg / day : 39% ( p = 0.01 ) ; 100 mg / day : 49% ( p < 0.001 ) ; 200 mg / day : 47% ( p < 0.001 ) ) exhibited a significant reduction in monthly migraine frequency than placebo - treated patients ( 23% ) . patients who received 200 mg / day tended to have more frequent adverse events.46 a third study took place in 88 neurology clinics throughout 21 countries in europe and the middle east . a total of 559 patients completed the 26-week open - label phase of the study and 514 of them who completed this phase continued to the 26-week randomized , double - blind , placebo - controlled phase of the trial . patients were assigned to topiramate ( n = 255 ) or placebo ( n = 259 ) . although the number of migraine days increased and the quality of life was lower , sustained benefit was observed after discontinuation of topiramate . the mean increase in number of migraine days before and after topiramate discontinuation was greater in the placebo group ( 1.19 days in 4 weeks , p < 0.0001 ) compared to the topiramate group ( 0.10 days , p < 0.5756 ) . the results of this trial suggest that patients should be treated for 6 months with the option to continue to 12 months in some cases.47 these pivotal trials are supported by a fourth study performed in the headache centre in rome , where 35 patients were assigned to topiramate and 37 patients to placebo . moreover we observed a significant reduction in the quantity of symptomatic drugs taken as compared to the placebo group ( from 6.17 1.80 to 2.57 0.80 ) and a significant downward trend in the number of days of disability.48 in a combined analysis , to piramate showed a greater reduction in migraine frequency than placebo . several large , randomized , placebo - controlled clinical trials have shown that topiramate administered at 100 mg daily significantly reduced the number of migraine headache days in patients with episodic migraine who experienced between three and 12 migraine episodes per month48,49 and in patients with chronic migraine who experienced 15 headache days per month.52,53 furthermore , the number of participants with a 50% or greater reduction in headache frequency with topiramate was significantly better than with placebo.49 in these clinical trials topiramate treatment was safe and generally well - tolerated . a double - blind randomized clinical trial compared zonisamide efficacy to topiramate in migraine prophylaxis . a total of 80 patients were recruited and randomly allocated to zonisamide 200 mg / day and topiramate 100 mg / day . zonisamide was gradually titrated up from 50 mg / day to 200 mg / day and topiramate from 25 mg / day to 100 mg / day . both drugs were associated with a significant decrease in frequency and severity of migraine along with a decrease in the need for acute medication in migraine attacks and migraine disability assessment score . no significant differences were observed between the 2 groups other than greater reduction in headache severity with zonisamide . these results suggest that zonisamide is as effective as topiramate in migraine prophylaxis and therefore can be considered as an alternative treatment in patients with poor tolerance to topiramate . larger studies are still needed before zonisamide can expect fda approval.1 two double - blind , randomized , placebo - controlled trials have shown that gabapentin reduces the frequency and intensity of migraine attacks.50,51 the first study was conducted during a period of 3 months , at seven participating centers . ninety eight patients were assigned to gabapentin and 45 to matching placebo . during the 4-week titration phase , patients were started on 300-mg / day gabapentin , increasing to 900 mg / day in the first week , and thereafter had weekly increases to 2400 mg / day . mean migraine frequency decreased significantly in the gabapentin treated group ( from 4.2 to 2.7 ) versus the placebo - treated patients ( from 4.1 to 3.5 ) . the number of patients achieving at least 50% reduction in monthly headache frequency with gabapentin was significantly better than with placebo ( 46% versus 16% , p < 0.01).51 in the second study , gabapentin was used at a dosage of 1200 mg / day and compared with placebo . it was associated with a significant reduction in the frequency and intensity of migraine compared with placebo.50 the primary measure of efficacy is the change in frequency of migraine attacks per month ( 28 days ) from the baseline frequency . results are considered clinically relevant if a reduction of 50% or more in migraine frequency can be demonstrated.46 preventive drugs must be used for periods of months . although there is no general agreement on the ideal duration of prophylaxis , recently published data suggest greater efficacy with longer treatment periods.52 valproate has been demonstrated to be an efficacious and well - tolerated agent for the preventive treatment of migraine , chronic daily headache and cluster headache and has received the approval of the fda.28 the recommended oral starting dose is 250 mg taken at bedtime and is gradually increased , usually by 125250 mg per week , to the desired dose of 750 mg per day in 23 divided doses.53 topiramate has fda approval for migraine prophylaxis.54 at a dose of 100 mg per day , it is reported to be the most effective and well tolerated of all drugs used for migraine prevention.55 a dose of 200 mg per day is no more effective than the 100 mg per day and is not as well tolerated . in the responders , positive effects were usually seen within the first month , and improvement continued during the 6-month observation period.43,49 the efficacy of topiramate was further increased with longer periods of prophylaxis . a group of migraine patients were treated for 8 months in an open - label extension phase after two large double - blind , placebo - controlled trials of 26 weeks duration . the mean number of attacks decreased from 3.4 2.6 per month at the end of the double - blind treatment periods to 2.2 2.4 per month after completion of the open - label extension phase with the active drug.56 zonisamide acts slower than topiramate , requiring 3 months to reach a 2/3 reduction in frequency of migraine attacks . it has been shown to be effective and well tolerated for migraine prevention in patients refractory to topiramate.34,57 a better control in headache severity was obtained with zonisamide in comparison to topiramate . these advantages can be explained by its specific mechanisms of action.1 gabapentin is less effective than topiramate or valproate and is therefore considered as drug of second choice.58 efficacy has been shown with a daily dose of between 1200 and 1600 mg.51 adverse effects leading to withdrawal of therapy are more likely to occur during the initial titration periods . it has been demonstrated that the drop - out rate is lower during long - term treatment extension phases than during initial treatment periods.55 in order to avoid or minimize side effects and therefore reduce the risks of withdrawal from treatment , the daily dose should be increased slowly over a period of at least 4 weeks or more , until targeted clinical benefits are achieved or until adverse effects interfere . if no side effects emerge and the desired clinical response has not yet been achieved , the dose can be increased providing the ceiling dose for the drug . the majority of migraine prophylactic drugs can cause tiredness or dizziness , therefore it is better to give them in the evening.19,58 it is important to always bear in mind contraindications to and the risk / benefit ratio of the drug for any patient . the most common side effects of valproate are tiredness , drowsiness , dizziness , weight gain , tremor , hair loss , skin rash and nausea . its use during pregnancy is contraindicated due to teratogenicity ( neural tube defects).52 long - term treatment with neuromodulators may alter the metabolism of sex hormones . use of valproate in women appears to be associated with a frequent occurrence of reproductive endocrine disorders characterized by polycystic changes in the ovaries , high serum testosterone concentrations ( hyperandrogenism ) and menstrual disorders.59,60 younger women seem to be especially vulnerable to the effects of valproate on serum androgen levels and they are more likely to develop polycystic ovary syndrome . the age of patients should therefore be considered while prescribing this medication.60 in males , it causes sexual dysfunction by decreasing follicle - stimulating hormone and luteinizing hormone.61 the endocrine effects of the new generation of neuromodulators have not yet been widely studied.59 topiramate is generally considered to be safe and well tolerated in migraine prophylaxis . in clinical trials , paresthesia is the most frequent of these , reported by half of patients.62 adverse events are usually mild or moderate in severity ; they are transient and decrease substantially over time . cognitive adverse events , including tiredness , psychomotor slowing , drowsiness , language difficulties , and difficulties with memory and concentration , arise in about 22% of patients treated with 100 mg per day of topiramate compared with 10% in those given placebo . these usually decrease in the second month of treatment and do not typically require discontinuation of the drug . unlike most other migraine - prevention drugs , topiramate is more likely to be associated with weight loss ( two of three patients ) than with weight gain . serious adverse events leading to treatment discontinuation were infrequent in clinical trials ( 2% ) . the risk of renal calculi is increased especially in patients with an underlying predisposition . the risk of secondary angle closure glaucoma is rare.43 despite being efficacious , some patients will not tolerate the adverse effects and hence will need an alternative.1 zonisamide is a well tolerated antiepileptic drug with a generous safety profile.63 it has as similar mechanism of action to topiramate , and similar , but lower incidence of side effects.57 gabapentin most commonly causes dizziness , tremor , somnolence , nausea and ataxia . the choice of drug should take into account comorbidities , and patient preferences in addition to the overt risk / benefit ratio of the medication.65 sometimes infrequent migraine attacks might be enough to impair quality of life such that the patient feels that commencing prophylaxis is warranted . hence , the decision to start regular prevention is a tailored , individual one that must take patient preference into account . physicians should understand patient treatment preferences , and help select the drug most suited to their needs.66 it is axiomatic that the patient should understand the reasons for starting preventive treatment and feel comfortable with the drug chosen . this increases the likelihood of adherence to the long - term preventive treatment plan.3 the factors influencing patient preference are likely to involve the following parameters : effectiveness , duration of relief , attack recurrence , ease of use , required doses , side - events , time to go back to normal functioning.67 the patient should be aware of the timing and extent of clinical benefit . many preventive medications take a minimum of 3 or 4 weeks for a therapeutic response at a particular dose , and maximum clinical effect might take another 2 to 3 months , during which time compliance is most important . the inefficacy of one drug does not necessarily mean that all migraine prophylactic drugs are ineffective . different drugs may have to be tried before one is identified as being really helpful in prophylaxis . it is also very useful to explain the potential side effects of these drugs in order to engage the patient in decision - making process and ensure compliance . the patients should be assured that the medications do not induce tolerance or addiction.19,58,68 unlike patient preference for acute migraine treatment , patient preference for prophylaxis has not been well studied . in one study evaluating patient preference for migraine prophylaxis , patients were asked to rate the following aspects of headache prevention : efficacy , speed of onset , out - of - pocket expenses , adverse events , formulation of therapy , the type of treatment and frequency of dosing . each patient also evaluated 12 different clinical scenarios , each containing a simulation of 2 hypothetical headache preventive treatments , wherein patients could choose product a , product b , or neither . patients were informed of each product s efficacy data ( 50% , 75% , or 100% of headache elimination ) , adverse events profile ( weight gain , concentration difficulty , and/or fatigue ) , and dosing frequency . patients were more likely to choose treatments with higher efficacy rates , fewer adverse events and less frequent dosing schedule . they also preferred treatment options with higher efficacy rates even if increased adverse events occurred or more frequent dosing was necessary.66 the selection a migraine prevention agent should be made on the basis of its efficacy , cost , potential adverse events , impact on quality of life , headache profile , patient preference , previous efficacious or unsuccessful treatments and any coexisting disorders.54 the european federation of neurological society ( efns ) guidelines aim to give evidence - based recommendations for the drug treatment of migraine attacks and prophylaxis . the level a recommendation corresponds to drugs of first choice with efficacy and safety well established in clinical trials . b drugs are second line , with evidence of efficacy but are either less effective or have more side effects than level a. level c drugs are third line , with only probable efficacy . based on these guidelines , valproate in a dose of at least 600 mg and topiramate in a dose between 25 and 100 mg are the two recommended first line agents for the prophylactic treatment of migraine with an a level of evidence . gabapentin in a dose between 1200 and 1600 mg with a grade c is a drug of third choice . migraine has a major socioeconomic impact , causing a considerable burden to the patients , their families and the society . it deeply affects the wellbeing and general functioning , not only during the acute attack , but also in terms of work performance , family and social relationships , and school achievement.70 the major goals of migraine prophylaxis are improving patients quality of life by reducing migraine frequency , severity , duration and disability . the results of the different randomized controlled clinical trials have proven that migraine prophylaxis with anticonvulsants such as valproate and topiramate is generally a safe and effective way of reducing the attack frequency and thus the burden of migraine . despite their good efficacy , some patients discontinued the prophylaxis with these agents because of clinically significant adverse events . with its low rate of side events and better tolerance than valproate or topiramate furthermore its long half - life permits once - daily dosing , may enable better patient compliance.57 antimigraine prophylaxis is still relatively underused . only 15%20% of all patients that fulfill the criteria for migraine prophylaxis treatment receive the appropriate treatment.71 the side - effects , compliance , and cost of prolonged treatment are important limiting factors . physicians should therefore be aware that treatment strategies for migraine prophylaxis require patient understanding and acceptance . all the drugs currently used in migraine prophylaxis have been discovered serendipitously without consideration of migraine pathophysiology . the next generation of prophylactic drugs will be developed on the basis of the recent understanding of migraine pathophysiology . the identification of new anti - migraine drug targets will hopefully lead to more effective and specific treatments with fewer side events . cortical spreading depression inhibition and calcitonin gene - related peptide antagonists such as talcagepant , seem promising , but need further investigation . drugs such as melatonin , vitamin e and botulinum toxins seem to have only marginal or no effect.72 migraine is a polygenic multifactorial disorder that is most likely influenced by multiple genes and environmental triggers.13 the emerging genetic findings will have implications for better understanding of migraine pathogenesis . they will hopefully lead to the development of new effective drugs with a better design and fewer side effects , and change the future of migraine therapy . the tresk channel represents an interesting target for the development of migraine specific treatment and needs therefore to be further explored . upregulation of this channel s activity could be of great benefit for migraine sufferers , either as in acute treatment or long - term prophylactic.4

this chapter will focus on the role of micrornas ( mirs ) in regulating the actions of opioid drugs through the opioid receptors . opioids , such as morphine , are analgesics that are used for treating many forms of acute and chronic pain . however , their chronic use is limited by undesirable effects such as opioid tolerance . the opioid receptor ( mor ) is the primary receptor responsible for opioids analgesia and antinociceptive tolerance . the long 3-untranslated region ( 3-utr ) of mor mrna is of great interest since this region may contain elements for the post - transcriptional regulation of receptor expression , such as altering the stability of mrna , influencing translational efficiency , and controlling mrna transport . micrornas are small non - coding rna molecules that exert their functions through base - pairing with partially complementary sequences in the 3-utr of target mrnas , resulting in decreased polypeptide formation from those mrnas . since the discovery of the first mir , lin-4 in caenorhabditis elegans , hundreds of mirs have been identified from humans to viruses , which have provided a crucial and pervasive layer of post - transcriptional gene regulation . the nervous system is a rich source of mir expression , with a diversity of mir functions in fundamental neurobiological processes including neuronal development , plasticity , metabolism , and apoptosis . recently , the let-7 family of mirs is found to be a critical regulator of mor function in opioid tolerance . let-7 is the first identified human mir . its family members are highly conserved across species in sequence and function . in the review , we will present a brief review of the opioid receptors , their regulation , and opioid tolerance as well as an overview of mirs and a perspective how mirs may interact with mor and serve as a regulator of opioid tolerance .

since first isolated from opium poppy in the early nineteenth century , morphine and related opioids remain as the most powerful analgesics to treat many forms of acute and chronic pain . however , repeated and prolonged use of opioids leads to tolerance , of which , the analgesic tolerance is the most problematic that can lead to therapeutic failure ( i.e. , inadequate pain control ; mcquay , 1999 ) . in some cases , even the highest tolerable dose of an opioid can not achieve the desirable analgesic effect in patients ( wang and wang , 2006 ; harden , 2008 ) . development of opioid tolerance not only limits the analgesic efficacy , but the increased doses of opioids in order to counter tolerance exacerbate another problem , namely opioid addiction that is a significant medical and public health problem . extensive efforts have been made to elucidate the mechanisms underlying opioid tolerance and drug addiction ( kieffer and evans , 2002 ) . increasing evidence implicates the contribution of transcriptional and epigenetic regulation in opioid tolerance and drug addiction , such as activation and inhibition of transcription factors ( carlezon et al . , 2005 ; zachariou et al . , 2006 ) , modification of chromatin and dna structure ( renthal et al . , 2008 ; guo et al . , 2011 ) , and induction of non - coding rnas including micrornas ( pietrzykowski , 2011 ; robison and nestler , 2011 ) . micrornas ( mirs ) are small non - coding rna molecules that repress target gene expression through base - pairing with partially complementary sequences in the 3-untranslated region ( 3-utr ) of target mrnas . owing to recent cloning , sequencing , and computational efforts , the numbers of known mirs has been rapidly increasing , and to date , there are a total of 21,643 mature mirs found across 103 species , of which 1921 mirs are found in humans ( mirbase release 18.0 , november 2011 ) . with the emerging identification of mirs from humans to viruses , a crucial and pervasive layer of post - transcriptional gene regulation by mirs has been elucidated ( ambros , 2004 ; taft et al . , 2010 ) . it is generally accepted that mirs serve as an important class of epigenetic regulators that participate in a variety of cellular activities . in respect to their diverse functions , mirs play an integral role in fundamental neurobiological processes including neuronal development , plasticity , metabolism , and apoptosis ( kosik , 2006 ) . as one form of long - lasting synaptic plasticity , opioid tolerance is a particularly interesting research area to study mir - mediated cellular adaptation . in this review , we summarize recent findings on mirs in opioid tolerance , with a focus on the role of let-7 mirs in regulating opioid tolerance . the opioid receptor ( mor ) , a member of the g - protein coupled receptor superfamily , is the primary receptor responsible for opioids analgesia and antinociceptive tolerance ( matthes et al . , 1996 ; sora et al . , 1997 ) . opioid tolerance may be result from opioid receptor desensitization and trafficking , which include opioid receptor down - regulation , internalization , and uncoupling from g - proteins due to chronic exposure to opioid agonists ( bailey and connor , 2005 ; martini and whistler , 2007 ; koch and hollt , 2008 ; lopez - gimenez and milligan , 2010 ) . receptor down - regulation as one of mechanisms contributing to opioid tolerance has been previously proposed ( davis et al . , 1979 ; tao et al . , 1987 ; bhargava and gulati , 1990 ; bernstein and welch , 1998 ; diaz et al . , 2000 ) . chronic morphine treatment produced a marked decrease in brain mor density ( davis et al . down - regulation of the high - affinity mor site in rats has also been reported following continuous infusion of morphine ( i.t . ; wong et al . , 1996 ) or etorphine ( s.c . morphine - induced mor down - regulation was also observed in sh - sy5y cells , with or without differentiation ( zadina et al . in addition to receptor down - regulation , chronic treatment with morphine has been shown to significantly reduce mor - signaling in sensory neurons and brainstem nuclei ( sim et al . , 1996 ; johnson et al . , 2006 ) , which are in agreement with the findings that reduced receptor number and resultant reduced mor - signaling on the other hand , there have been reports that mor expression was not altered ( dum et al . , 1979 ) or even up - regulated ( lewis et al . , 1984 ) in the brain by various opioids . some of the discrepancies may be caused by uncontrolled variables ( e.g. , different opioids , doses , methods of opioid treatments , anatomical regions , and times samples were taken , integrality of tissue samples before assays , and opioid receptor subtypes studied ) , as well as detection methods . it has been suggested that mor down - regulation is agonist selective and depends on the agonist s intrinsic efficacy ( nishino et al . , 1990 ; the purity and selectivity of radiolabeled ligands a problem not only for some early studies , but also in more recent reports employing questionable materials used in studies are the other potential culprits for conflicting findings . the long 3-utr of mor mrna ( ide et al . , 2005 ; han et al . , 2006 ) suggests that this region may contain physiologically relevant elements for regulating receptor expression by mechanisms such as mir targeting . indeed , early research on 3-utr of human mor mrna suggested that mor expression was increased after a 712-bp segment , immediately downstream of the stop codon , was removed ( zollner et al . , 2000 ) . comparative bioinformatics predicted potential mirs that may interact with the human and mouse mor ( table 1 ) . let-7 family of mirs was identified as a top candidate according to the number of putative target sites and alignment pattern . our group experimentally validated the in silico prediction that members of the let-7 mir family can interact with the 3-utr of mor mrna at the predicted positions ( he et al . , 2010 ) . furthermore , downregulating let-7 with specific lna - modified antisense oligodeoxynucleotides ( lna - let-7-as ) was found to increase mor expression in sh - sy5y cells , a human neuroblastoma cell line , suggesting that ( 1 ) mor is a target of let-7 ; ( 2 ) expression of mor is under constitutive suppression by let-7 . microrna targets predicted by miranda ( http://www.microrna.org/microrna/getgeneform.do ) and ranked according to the alignment scores . in order to elucidate a physiological role of let-7 in regulating mor and opioid tolerance , we further examined the in vivo relevance of let-7 mirs in cellular and animal models of opioid tolerance . for the former , sh - sy5y cells were treated with morphine ( 110 m for 2448 h ) to induce cellular tolerance . the expression of let-7 mirs was significantly up - regulated by chronic treatment with morphine , while the expression level of mor was reduced as determined by the western blotting method ( he et al . , 2010 ) and the receptor radioligand binding using h - damgo ( he and wang , unpublished data ) . therefore , chronic morphine treatment caused the increase of let-7 and decrease of mor expression during the development of opioid tolerance in sh - sy5y . furthermore , the regulation of let-7 expression by morphine occurred in a mouse model of opioid tolerance . mice were implanted s.c . with a 75-mg morphine pellet ( 75 mg morphine pellet / mouse , s.c . ) . brain expression of let-7 increased gradually over time after morphine treatment , temporally correlating with the development of antinociceptive tolerance to morphine . interestingly , up - regulation of let-7 occurred in mor - expressing cells , but not in mor - negative cells in mice brain cortex region as determined by in situ hybridization ( he et al . , 2010 ) . this was in agreement with the aforementioned finding that mor is a direct target of let-7 . in order to further examine a causative role of let-7 in leading to opioid tolerance , we directly targeted let-7 in the mouse model of opioid tolerance . treatment with the let-7 inhibitor ( i.c.v . ) decreased brain let-7 levels and partially attenuated opioid antinociceptive tolerance ( he et al . , 2010 ) . previous reports from a number of different cell lines or animal models ( e.g. , brodsky et al . , 1995 ; johnson et al . , 2006 ) indicated that mor mrna was not changed upon treatment with morphine . for example , mor mrna remained the same in sh - sy5y cells following chronic morphine treatment . we confirmed that let-7 did not affect mor mrna stability ; however , polysome - bound mor transcript was significantly decreased . so these intriguing data led us to propose the following regulatory mechanism where upon let-7 can regulate opioid tolerance : let-7 recruits and sequesters mor mrna to p - bodies that are deprived of translational machinery , effectively reducing polysome - bound mor mrna and leading to translation repression . a similar pathway was observed in hek293 and hela cells ( pillai et al . , 2005 ) , thus translation repression may serve as a general mechanism by let-7 to regulate its target gene expression ( figure 1 ) . keep in mind that let-7 activity is turned up in opioid tolerance by up - regulation of their transcripts . nature works in a wonder to ensure the activity of let-7 , ultimately dampening the activity of opioids upon persistent activation . a schematic diagram proposing a mechanism for the regulation of target gene by let-7 . pri- and pre - let-7 are produced and exported into the cytosol where the mature let-7 was incorporated into the rna - induced silencing complex ( risc ) . the latter recruits and sequesters target mrna to p - bodies that are deprived of translational machinery , effectively reducing polysome - bound mrna and leading to translation repression ( modified from he et al . , 2010 ) . its family members are highly conserved across species in sequence and function ( pasquinelli et al . , 2000 ) . major roles of let-7 include the regulation of stem - cell differentiation , neuromuscular development , and cell proliferation & differentiation ( reinhart et al . , 2000 ; let-7 was initially identified as a heterochronic gene ( pasquinelli et al . , 2000 ) . in mammals , let-7 levels increase during embryogenesis and during brain development ( schulman et al . , 2005 ; let-7 is undetectable in human and mouse embryonic stem cells , and the level of let-7 increases upon differentiation ( thomson et al . , 2004 , 2006 ; this high expression of let-7 is then maintained in various adult tissues ( sempere et al . furthermore , let-7 is widely viewed as a tumor suppressor mir ( boyerinas et al . , 2010 ) . there is a clear link between loss of let-7 expression and the development of poorly differentiated , aggressive cancers ( takamizawa et al . , 2004 ; 2009 ) . using the computational algorithm targetscan 6.0 to screen human 3-utr sequences containing let-7 family mirs complementary sites , 886 conserved targets , with a total of 989 conserved sites and 111 poorly conserved sites were predicted . high - mobility group at - hook 2 ( hmga2 ) , which is the top - scoring candidate gene on the list ( table 2 ) , was confirmed as a direct let-7 target both in vitro and in vivo ( lee and dutta , 2007 ; mayr et al . hmga2 is a chromatin - associated non - histone protein capable of modulating chromatin architecture and thus affecting transcription . in addition to abundant studies on hmga2 in embryogenesis ( zhou et al . , 1995 ; sun et al . , 2009 ) and tumorigenesis ( peng et al . , 2008 ; qian et al . , 2009 ) , it would be interesting to investigate its possible involvement in opioid tolerance as a functional target gene of let-7 . top 100 predicted targets of let-7 mirnas ranked by total context score ( targetscan 6.0 , http://www.targetscan.org/. ) seed pairing rules are widely used to predict functional mir target sites . mir - mrna recognition requires the perfect complementarity of 6-nucleotide mrna seed sites with the 5 end of mirs ( positions 27 ; bartel , 2009 ) . however , when predictions based on such short complementary sequences are compared to experimental results from proteomic studies , the false - positive and false - negative rates appear to be above 50% ( easow et al . , 2007 ; baek et al . , 2008 ; mourelatos , 2008 ; selbach et al . , several previous findings strongly indicated that a sizeable fraction of mir targets do not obey the canonical seed rule ( ha et al . , 1996 ; didiano and hobert , 2006 ; tay et al . , 2008 ; stefani and slack , 2012 ) . for let-7 , biological studies clearly demonstrated that genetically verified let-7 targets in caenorhabditis elegans contain only imperfect binding sites , with bulges or gu wobble pairs in the seed region ( vella et al . , a recent study identified a new class of mir target site nucleation bulges and an alternative mode of mir target recognition by a pivot - pairing rule ( chi et al . , 2012 ) . they proposed a transitional nucleation model in which a transitional nucleation state determines the binding of mirs to nucleation bulge mrnas . therefore , the identification of non - canonical let-7-mrna interactions may lead to important breakthroughs in discovering new let-7 targets and further decipher the functional role of let-7 in opioid tolerance . with respect to let-7 target gene identification , another important issue need to be addressed is the redundancy of let-7 family members . there are 14 and 13 different let-7 family members in mouse and human , respectively ( roush and slack , 2008 ) . in human , these different members , let-7a-1 , 7a-2 , 7a-3 , 7b , 7c , 7d , 7e , f7 - 1 , 7f-2 , 7 g , 7i , mir-98 , and mir-202 are located on nine different chromosomes ( ruby et al . , 2006 ) . while let-7 was initially viewed as one single activity , emerging data suggest that the let-7 family contains mirs with different activities ( i.e. , targets ) . for example , it has been reported that let-7b was highly expressed but let-7e was drastically reduced in malignant mesothelioma ( guled et al . , 2009 ) . so the question remains as whether individual let-7 family member with its own expression pattern exerts specialized function during opioid tolerance development . opioid tolerance , even to the analgesic actions of these drugs , is likely not caused by a single mechanism ; rather an intricate neuronal circuitry involving multiple mechanisms may ultimately lead to the expression of tolerance seen in humans . regulation of opioid tolerance , through mor or other mechanisms , represents one of these mechanisms . a typical study in mir research field tends to survey transcript levels of mirs in a disease state ( zheng et al . , 2010 ) . for example , morphine - induced up - regulation of mir-15b in human monocyte - derived macrophages ( dave and khalili , 2010 ) , while mir-133b was decreased by morphine in zebrafish embryos ( sanchez - simon et al . , 2010 ) . furthermore , moving from a cell line to in vivo relevance is another big hurdle to overcome . in the case of let-7 mirs , it was identified as a critical regulator of both human and mouse mor . moreover , it was demonstrated to be relevant for both cellular opioid tolerance as well as animal models of opioid tolerance ( he et al . , 2010 ) . let-7 represents one of several mirs contributing to opioid tolerance . for example , it was found mir-23b could interact with the mor 3-utr via a k box motif ( 5-ugugau-3 ) in sh - sy5y and mouse p19 cells ( wu et al . , 2008 , 2009 ) . while mor is involved in the development of morphine tolerance , it is not the only target for the action of mirs in opioid tolerance ( matthes et al . , 1996 ; sora et al . , 1997 ) further research is needed to identify other target genes of mirs ( including let-7 ) . a number of receptors , ion channels , and protein kinases , which have been implicated in opioid tolerance , such as the nmda receptors , pkc , and camkii ( kieffer and evans , 2002 ; wang and wang , 2006 ; ueda and ueda , 2009 ) may become potential targets for let-7 . in summary , mir - mediated mechanisms provide a novel direction toward unraveling the complex mechanisms involved in the development of opioid tolerance . these studies will enrich our knowledge on basic principles of neuronal and behavioral adaptation in opioid tolerance , and eventually lead to novel design and development of pharmacological treatments for opioid tolerance . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

introduction : dentine hypersensitivity is characterized by acute , sharp pain arising from the exposed dentine , most commonly in response to thermal , tactile , or chemical stimuli , and which can not be linked to any other pathological changes in the tooth or the environment . therapy uses various impregnating agents in the form of solutions or gels and , in more recent times , laser.aim:the aim of this research was to examine the effects of treatment of hypersensitive dental cervix with diode laser.materials and methods : the study included 18 patients with 82 sensitive teeth . the degree of dentine hypersensitivity was evaluated by visual analogue scale ( vas ) , and the treatment was carried out by application of low - power diode laser over the span of three visits , which depended on the initial sensitivity.results:there is a significant difference in vas values measured at the onset of treatment ( baseline ) and immediately after the first laser treatment ( t=9.275 ; p=0.000 ) , after 7 days , after the second laser treatment ( 14 days ) ( t=7.085 , p=0.000 ) , as well as after 14 days and the third laser treatment ( t=5.517 , p=0.000 ) , which confirms the effectiveness of this therapeutic procedure . the results showed a reduction of hypersensitivity in response to tactile stimulus with a probe after the third treatment , even with teeth whose value on the vas was very high at the beginning of treatment ( baseline).conclusion : within the scope of the conducted study , laser therapy has provided extremely safe and effective results in the treatment of cervical dentine hypersensitivity .

thanks to good preventive dental programs and developed knowledge about the importance of oral hygiene , the vitality of teeth within the oral cavity has been extended in the recent times , which led to an increase in non - carious cervical lesions , or dental erosions , abrasions , etc . dentine hypersensitivity ( dh ) is characterized by acute , sharp pain in the area of exposed dentine , in response to thermal , chemical , osmotic , and tactile stimuli ( 3 ) . although sensitivity can occur on any part of the tooth , it is most commonly felt in the vestibular area of dental cervical region ( for canines and first premolars ) and on the root surface . the frequency at which it occurs ranges between 3 and 57% , and it is much more frequent ( 72 - 98% ) in patients suffering from periodontal disease . it most often occurs between 20 and 50 years of age , and is more common among women ( 4 , 5 ) . difficulties in treating cervical dh gave rise to a large number of techniques and therapeutic procedures which are currently used for pain alleviation in dh ( 6 ) . therapy uses various impregnating agents in the form of solutions or gels and , in more recent times , laser . based on hydrodynamic theory , several methods , such as the application of fluoride , dentine adhesives , corticosteroids , and silver nitrate solution , work by blocking the open dentine tubules . number of treatments compared to average vas value at baseline ( at the beginning of treatment ) baseline assessment of tooth sensitivity and number of treatments in recent decades , classic treatments with desensitizing agents have been supplemented by the use of laser . using lasers to treat dh dates back to the 80s with the advent of erbium lasers . even though the initial results were quite disappointing , the improvement of technology and scientific knowledge over time has led to the development of new lasers with wavelengths suitable for therapeutic treatment ( 7 ) . most studies conducted with different types of lasers , with different wavelengths and duration of application , reveal the effectiveness of this treatment , both immediately upon the completion of therapy , and after circa 6 months from the first treatment . as a result , the pain is reduced and , in many cases , eradicated ( 8 , 9 , 10 ) . among the published works , there are those which confirm the exceptional efficacy of the use of diode lasers in the treatment of dh . thus , the aim of our study was to investigate the effects of diode laser therapy on hypersensitive dental cervix . dentine hypersensitivity was stimulated by touching the dental cervix with the tip of the probe , with mesial - distal directionality . all patients were asked to assess their level of dentine hypersensitivity using the vas scale of 0 to 10 , where 0 represents no pain and 10 represents greatest pain . after initial sensitivity was assessed and recorded , laser therapy was initiated . laser treatment protocol : low - power diode laser ( smilepro980 , biolitec ) was used in this study . the laser was operated in a continual regime , and 2 w of power was applied to the tooth surface . during the 60 seconds of exposure , tooth tissue was around 2 mm away from the laser . exposure time ( 60s ) was repeated after sensitivity control ( using the vas scale ) , seven and fourteen days after initial exposure , only on those teeth that were still sensitive . while working with the laser , both the therapist and the patient wore protective goggles , and work space the study included 18 patients , with average age of 27 years , who had 82 sensitive teeth . there is a significant difference in tooth sensitivity values measured at baseline , in teeth that had a different number of laser treatments . , we can say ( with 95% confidence ) that teeth which had lower dentine sensitivity at the very beginning will require fewer laser treatments . in order to determine between which teeth this difference is observed , given the number of treatments , a post - hoc analysis was carried out using turkey s honest significant difference ( hsd ) test . differences occur only between the mean sensitivity values at baseline between teeth that had only one treatment and teeth that had three laser treatments ( p=0.037 ) , but there is no difference between vas value at baseline between teeth that had one laser treatment and those that were treated twice ( p=0.073 ) , nor between the teeth that had two and those that had three laser treatments ( p=0.934 ) . there is a significant difference in vas values measured at baseline and after the first laser treatment : t=9.275 , p=0.000 . there is a significant difference in vas values measured at baseline and after the second laser treatment : t= 1.268 , p=0.000 . there is a significant difference in vas values measured at baseline and after the third laser treatment : t=8.749 , p=0.000 . vas values of all teeth before treatment and following the first , second , and third laser application vas values for all teeth after the first , second , and third application of laser , and baseline measurement prior to the application of laser there is a significant difference in vas values measured at baseline and after the first laser treatment ( t=9.275 ; p=0.000 ) , as well as after 7 days and after the second laser treatment ( 14 days ) ( t=7.085 , p=0.000 ) , and after 14 days and the third laser treatment ( t=.517 , p=0.000 ) , which supports the effectiveness of this therapeutic procedure . dentine hypersensitivity ( dh ) is common , and individual needs for treatment depend on aetiology , as well as on the subjective experience of painful sensations and the degree of tolerance to this type of pain . in this study , some of the patients reported pain so severe that it has become a physical and emotional problem that affects their quality of life . many of them were not able to consume hot or cold foods or liquids , acidic foods or liquids , and even had difficulty with brushing teeth . as the data from previous studies suggest , several methods should be applied during treatment in order to obtain satisfactory results , since the aetiology of dh may be multifactorial ( 11 , 12 , 13 ) . conventional methods of treating dh include topical application of desensitizing agents , either professionally or at home , such as protein precipitates , agents for occlusion of dentinal tubules ( 14 ) and , more recently , lasers ( 15 , 16 , 17 ) . it is believed that the occlusion of dentinal tubules leads to a decrease in permeability of dentine and , proportionally , also reduces dh ( 18 ) . according to hydrodynamic theory , efficacy of dentine desensitization agents is directly related to their ability to efficiently close dentinal tubules ( 19 , 20 ) . in their study , yilmaz et al . compared the effectiveness of application of sodium fluoride and diode laser in the treatment of dh . they concluded that , within the scope of their study , gaalas laser therapy is effective in the treatment of dh , and is a more comfortable and faster treatment than traditional treatments for dh ( 21 ) . several studies ( 22 , 23 ) describe the synergistic effect of lasers in conjunction with desensitization agents . for this reason , our study included laser irradiation of the cervical portion of the tooth only , and we obtained exceptionally good results in terms of lowered dentine hypersensitivity ( f = 3.77 , p = 0.027 ) . therefore , we can state ( with 95% confidence ) that teeth which had lower dentine sensitivity at the very beginning will require fewer laser treatments . previous published data indicate that only the nd : yag laser has an additional analgesic effect , compared with other lasers . these findings are the result of the effect of radiation which can temporarily alter the endings of sensory axons and block both c and ab fibres , thereby reducing the pain ( 24 ) . parameter of the power used in our study was 2 w , which is in accordance with the study by liu et al . their study ( 25 ) demonstrated that 2 w ( 166 j / cm ) is a suitable parameter for the 980 nm diode laser , which sealed dentinal tubules without excessive melting of the dentine , thus achieving a good level of analgesia , which is comparable to our results . good results arise from the closure of dentinal tubules , which prevents internal communication of dental pulp with oral cavity fluids ( 15 , 26 ) . based on the results of our study in which only a diode laser was used , we believe that modern low - power lasers can also provide good results in the treatment of dh ; this finding is also supported by the results of research by umberto et al . ( 27 ) . our research , as well as research by other authors ( 28 , 29 ) , demonstrates that low - energy lasers , including gaalas diode laser with wavelengths between 780 and 980 nm , have an effect on nerve endings , thus eliminating sensitivity . in a study conducted on 27 patients with 55 hypersensitive teeth , lopes et al . assessed the efficacy of various protocols for treating dentine hypersensitivity . they concluded that all desensitising protocols are effective in reducing dentine hypersensitivity , but have different effects . therefore , they believe that a combination of protocols is an interesting alternative for the treatment of cervical dentine hypersensitivity ( 30 ) . this conclusion follows from the need to achieve satisfactory results in as few treatments as possible . the results of our study indicate that , applied through multiple treatments , this modern therapeutic procedure independently achieves good results , even in teeth with greater level of hypersensitivity . we believe that further research is needed to assess long - term effects of these therapeutic procedures on a larger sample in order to provide recommendations for use in routine clinical practice . within the scope of the conducted study , laser therapy has provided extremely safe and effective results in the treatment of cervical dentine hypersensitivity .

background and objectives . the purpose of the present study is to find the genes and snp that influence bmd and postmenopausal saudi women . material and methods . two - hundred ethnic saudi arabian women with a diagnosis of postmenopausal osteoporosis were the subjects of this study . baseline blood hematology , biochemistry , and bone panel were done . blood was collected , and three taqman - mgb probes were used to analyze snp variants in alox15 ( rs7220870 ) , lrp5 ( c 25752205 10 ) , and tnfrsf11b ( c 11869235 10 ) . results . the variant of alox15 17p13 showed that the bmd of the spine was lower in the aa allele ( p value < 0.002 ) and fractures were highest at 50% compared to cc allele . in the tnfrsf11b gene , bmd of the hip and spine was significantly higher in the gg allele and the history of fractures was significantly higher in gg group . with regard to the lrp5 ( c 25752205 10 ) gene , there was no significant difference between allele groups . conclusion(s ) . this study shows that the genetic influence of osteoporosis in the caucasian and saudi arabians population is similar . we believe that the same genetic markers that influence osteoporosis in the caucasian race could be used for further studies in the saudi arabian population .

postmenopausal osteoporosis ( pmo ) has become a major epidemic of the last millennium and is expected to be a problem for health care providers in the present millennium as well . osteoporosis is a disease in which the net loss of bone exceeds bone formation , and it occurs in women after estrogen loss in postmenopause [ 13 ] . postmenopausal osteoporosis ( pmo ) is a major public health epidemic worldwide . most women with pmo present with a fracture as the first indication of the disease . it is estimated that , in usa , 25 million women suffer from osteoporosis , and the cost to treat osteoporosis - related fractures ( orfs ) exceeds $ 10 billion a year [ 5 , 6 ] . the prevalence of pmo in saudi arabia and the recognition of the problems associated with pmo were only realized in the last decade . only few studies about pmo in saudi arabian women have been reported [ 710 ] . the most serious complication of osteoporosis is hip fracture , which increases patients ' morbidity and mortality rates . the incidence and costs of these fractures and their sequelae will continue to rise as the population ages ; by year 2025 , costs related to osteoporotic fractures are projected to reach $ 25.3 billion in the united states alone . in an assessment of the annual cost of orf in saudi arabia , bubshait and sadat - ali ( 2007 ) found that sr 4.27 billion is spent yearly to treat osteoporosis - related femoral fractures . osteoporotic hip fracture usually requires hospitalization and surgery and may result in lengthy or permanent disability or even death . within the first year after injury , a patient with a hip fracture has a 1015% greater chance of dying than others of the same age . men , though suffering fewer hip fractures than women , are 25% more likely than women to die within one year of the injury [ 1315 ] . from family histories , twin studies , and molecular genetics , it is quite evident now that some of a patient 's predisposition for osteoporosis can be inherited . genetic control of osteoporosis is polygenic ; enumeration of the specific genes involved is in the initial phases [ 2 , 1618 ] . there is no study , as yet , about the genetic influence on osteoporosis and related fractures in saudi arabian women . this study is conducted to find the influence of known genes on osteoporosis among saudi arabian women with and without fractures . after approval of the ethical and review board of the university of dammam and written consent from the patients , two - hundred ethnic saudi arabian women with diagnosis of postmenopausal osteoporosis by dexa machine on the basis of t and z scores , as described by who , were the subjects of this study , which was done between january 2009 and june 2010 . baseline blood hematology , biochemistry , and bone panel were also done . for the genetic analysis , 5 ml of whole blood was collected . out of several candidate osteoporosis genes which have been reported in the caucasian race to influence bmd and fragility fractures , we did genotyping of 3 polymorphisms in three genes as a pilot study in ethnic saudi arabian postmenopausal women . the three genetic polymorphisms have been convincingly shown to affect the bmd and fragility fractures in the white race . three taqman - mgb probes ( two predesigned and one on design assays ) have been used to analyze snp variants in the requested genes : alox15 ( rs7220870 ) , lrp5 ( c_25752205_10 ) , and tnfrsf11b ( c_11869235_10 ) . dna from 100 l blood samples has been extracted using the dneasy tissue extraction kit from qiagen . positive dna control of known genotype ( wild type , heterozygote , and mutant ) was added to the pcr as well as negative control ( no dna ) . genotyping was visualized on taqman 7000 ( applied biosystems ) . the data of the patients including age , history of fractures , and hip and spine bmd was entered and analyzed using spss inc . 's statistical package for social sciences ( spsss ) , version 14.0 ( chicago , il , usa ) . p values less than.05 and a ci of 95% were used to indicate statistical significance . the average age of the patients was 62.5 5.9 years . the variant of alox15 17p13 ( rs7220870 ) , c > a snp where c is the ancestral allele ( freq . 0.10.3 ) . majority ( 69% ) of the women were cc ( homozygous for the c allele ) ; even though bmd in the hip region was significantly lower ( p < 0.001 , ci 0.017 to 0.06 ) , history of fractures were lower than the other two alleles ( p < 0.0002 , ci 3.4 to 15.7 ) table 1(a ) . the number of women in the homozygous for a allele was 4% with a higher bmd . a history of fractures was found in 50% of the patients ( p < 0.001 ) ( table 1(b ) ) . regarding the analysis of the lrp5 rs3736228 , the alleles were c > t snp where c is the ancestral allele ( freq . 0.70.9 ) and the t allele is the variant allele ( freq . 0.10.3 ) . over 96% of the patients exhibited the cc and ct alleles . in women with the homozygous t allele the bmd was significantly higher at the hip and spine and there were no fragility fractures . the statistical analysis of the lrp5 gene and snp is given in table 2(b ) . in the tnfrsf11b ( c_11869235_10 ) gene , the majority of patients belonged to the aa and ag alleles , which was homozygous and heterozygous for the a allele , and only 3% belonged to the gg allele . bmd of the hip and spine was significantly higher in the gg allele , but the history of fractures was more common in the gg group p < 0.001 ( ci 59.85 to 60.146 ) ( tables 3(a ) and 3(b ) ) . in the a allele group , there was risk of low bmd and higher risk for osteoporosis without fractures , whereas , in patients with the g allele , there was higher bmd of both hip and spine and higher prevalence of fractures . our study provides evidence that earlier reported snps in the alox 15 , lrp5 , and tnfrs11b , which influences bmd , osteoporosis , and fragility fractures in caucasians , also influences the condition in ethnic saudi arabian females . . found that the t / t genotype of alox15 had a 33% higher rate of osteoporosis fractures . in this study we found that although only 4% of the women carried the genotype , the fracture rate in those who carried it was 50% . , mullin et al . reported that polymorphisms in alox15 are not associated with influencing bmd in white men and women . low - density lipoprotein receptor - related protein 5 ( lrp5 ) was reported to influence bmd and risk of fractures . variations of lrp5 have been linked to bmd and susceptibility to osteoporosis , but koller et al . suggested that lrp5 was not a major influence on attainment of peak bmd of the hip and spine in white women . reported that their findings indicate that lrp5 does influence bmd and fracture risk throughout life in the general population . mizuguchi et al . found that japanese women with the c / c genotype had higher adjusted bmd ( adjbmd ) value compared to those with c / t and t / t ( p = 0.022 ) , but in saudi women the c / c genotype had the lowest bmd in the hip and spine , and women with the tt allele had no fragility fractures compared to cc and ct variants . . meanwhile , found no significant difference in bmd between the three alleles in white men . the distribution of cc , ct , and tt alleles in this study was 73.5% , 23% , and 3.5% and similar frequency . the cc , ct , and tt genotypes were found in 75.6% , 21.8% , and 2.6% of the participants , respectively . . reported strong evidence of association with snp in rs4355801 on chromosome 8 , near tnfrsf11b , along with bmd and increased risk of osteoporosis . they found that the g allele at rs4355801 was associated with higher bmd , and the a allele with low bmd , while in our patients the bmd , was significantly higher with g alleles as compared to aa or ag alleles ( p < 0.001 ; ci < 0.1966 ) and lower in aa or ag alleles . even though the risk of osteoporosis was higher in patients with the a allele , the fracture prevalence was not increased . our results concur with the findings of richards et al . with regard to the risk of fractures with the a allele . the number of osteoporotic patients was small , compared to many studies on genetic analysis . since this is a pilot and comparative study , one can draw reasonable conclusions about the genetic influence on osteoporosis among the ethnic saudi arabian population . as many researchers resort to gwas to get minimal new data , we opted to study the influence of reported target osteoporotic genes upon the white race . in conclusion , there is strong similarity of the snps in the genes that influence bmd , osteoporosis , and fragility fractures among the reported snps and genes in the caucasian race and the ethnic saudi population . these may not be the only snps which influence osteoporosis risk , but it gives a direction to study the reported targeted genes among the saudi population .

purpose : to evaluate the effects of vitrectomy and internal limiting membrane peeling for treatment of macular edema secondary to retinal vein occlusion ( rvo).methods : nine cases of visual loss due to macular edema caused by central retinal vein occlusion or branch retinal vein occlusion underwent pars plana vitrectomy with removal of the preretinal hyaloid , peeling of the internal limiting membrane stained with indocyanine green dye , air fluid exchange , and postoperative prone positioning . best - corrected visual acuity ( bcva ) and central foveal thickness by optical coherence tomography were measured pre- and postoperatively then compared to assess the outcome of surgery.results:in all cases intraretinal blood and retinal thickening diminished within 2 months of surgery . visual acuity improved in all of the central retinal vein occlusion cases and 3/6 branch retinal vein occlusion cases . the decrease in macular thickness was statistically significant ( mean postoperative macular thickness 361 61.1 versus mean preoperative macular thickness 563.9 90.0 , p = 0.001 , t - test ) . the improvement in bcva was not statistically significant ( mean preoperative bcva in logmar 1.23 0.29 versus mean postoperative bcva in logmar 1.06 0.49 , p = 0.09 , t - test).conclusion : in eyes with macular edema secondary to rvo , pars plana vitrectomy with internal limiting membrane peeling can resolve macular edema , but the improvement in bcva was not statistically significant in this study .

retinal vein occlusion ( rvo ) is the second most common retinal vascular disorder after diabetic retinopathy and is a significant cause of visual morbidity . in these patients the development of me followed by rvo has been hypothesized to be caused by fluid flux from vessels to tissue according to starling s law,1,2 which is based on the breakdown of the blood retinal barrier as a result of damage to the tight junctions of capillary endothelial cells,3 vitreoretinal adhesion,4 and secretion of vasopermeability factors produced in the retina into the vitreous.5,6 observations by noma et al suggest that in patients with rvo , vascular occlusion induces the expression of vascular endothelial growth factor ( vegf ) and interleukin-6 ( il-6 ) , resulting in breakdown of the blood retinal barrier and increased vascular permeability.6 thus , vegf and il-6 may contribute to the development and progression of vasogenic me in rvo . me is closely associated with retinal hypoxia , and the degree of hypoxia in the center of the macula corresponds to the decrease in visual acuity ( va ) . if marked hypoxia persists , irreversible structural changes in the macula occur , and the disturbed va is permanent . if no spontaneous improvement occurs , treatment is mandatory to decrease the duration of edema and prevent photoreceptor damage . gutman et al found that that chronic me has a poor prognosis in terms of final va . longstanding me often results in permanent cystic macular changes.7 treatment modalities for me in rvo are laser treatment , intravitreal and periocular application of steroids , intravitreal injection of vegf inhibitors , radial optic neurotomy , and vitrectomy techniques . the present study looked at a new surgical treatment , based on previous hypotheses about the role of the vitreoretinal interface in the pathogenesis of me , and the popularity of vitrectomy techniques in recent years for the management of me . mandelcorn et al reported promising results with macular decompression using vitrectomy and internal limiting membrane ( ilm ) peeling in cases of severe visual loss from me due to rvo.8 they postulated that removal of the ilm in association with vitrectomy in these cases may allow the edematous retina to decompress by dispersion of retained intraretinal extracellular fluid and blood into the vitreous cavity through the inner retinal surface from which the ilm has been removed . this report describes the results of macular decompression by vitrectomy and ilm peeling , which was performed in the authors first nine consecutive cases of severe visual loss due to me in rvo . this study was a prospective nonrandomized clinical series including a total of nine eyes in nine patients . patients were known cases of crvo and brvo with me as confirmed by optical coherence tomography ( oct ) . inclusion criteria were onset of disease between 3 and 9 months before surgery and best - corrected visual acuity ( bcva ) of < 20/40 . exclusion criteria were any history of macular photocoagulation , any history of anti - vegf or corticosteroid injections in the vitreous and any concurrent pathology of the eye that decreased vision . all the surgeries were performed by the same surgeon . if there was no spontaneous posterior vitreous detachment , the preretinal vitreous cortex was removed from the retinal surface ( posterior - vitreous - detachment formation ) . after air fluid exchange , injection of 0.1 ml of 2.5 mg / ml indocyanine green dye ( icg ) solution ( akom , inc , buffalo grove , il ) was made into the vitreous cavity over the macula and allowed to act for 30 seconds to stain the ilm . the icg - stained ilm was then grasped with vitreous forceps and peeled away from the inner retinal surface over the macular area to a point just outside the foveal avascular zone . an air fluid exchange was carried out with postoperative prone positioning of the patient for 48 hours so that egress of extracellular fluid and blood from the inner retina could be facilitated using a squeegee mechanism similar to pneumatic displacement of submacular hemorrhage.913 for each patient , the first postoperative evaluation was done 1 week after surgery to check for possible complications , and the last examination to evaluate the final outcome of surgery was performed 2 months postoperatively . in this last visit retinal thickness there were no complications during surgery or in the follow - up period . according to fundus fluorescein angiography ( ffa ) findings , the time from initial onset of rvo to surgical intervention ranged from 3 to 9 months ( mean 5.3 months ) . preoperative bcva ranged from 20/100 to 20/800 , and preoperative foveal thickness ranged from 411 to 701 m ( mean 563.9 90.0 ) . two months after surgery , the bcva of all crvo cases improved , but only half of the brvo cases gained better va . there was a statistically significant decrease of macular thickness ( postoperative mean macular thickness 361 61.1 , p = 0.001 , t - test ) . visual acuity improved in all of the crvo cases and three out of six brvo cases , but the improvement in bcva was not statistically significant ( mean preoperative bcva in logmar 1.23 0.29 versus mean postoperative bcva in logmar 1.06 0.49 , p = 0.09 , t - test ) . when ischemic cases were excluded , improvement of bcva was still not statistically significant ( p = 0.06 , t - test ) . the mechanism of visual loss in me due to rvo is thought to be a combination of interference with photoreceptor stimulation by a thickened , hemorrhagic , and edematous retina in association , in some cases , with ischemic retinal damage due to concomitant retinal ischemia.1419 it was hypothesized that vitrectomy with removal of the ilm would allow the congested , hemorrhagic retina to decompress by facilitating the release of extracellular fluid and blood into the vitreous , which would , in turn , restore normal retinal thickness and intraretinal tissue pressure , reduce opacities within the retina that could interfere with light transmission to photoreceptors , and reduce intraretinal pressure around adjacent retinal venules and capillaries that would allow these blood vessels to reopen where possible . furthermore , by performing a vitrectomy , sequestered cytokines such as il-6 and vegf can be removed from the eye , so their adverse effects can be eliminated . the vitrectomy performed during this intervention probably improved preretinal oxygen tension2,20,21 and , secondarily , may have decreased levels of vegf . removal of the ilm might have resulted in decompression of the me and a further improvement in preretinal oxygen tension.8,22 vasoconstriction stimulated by increased oxygen tension might have reduced hydrostatic vessel pressure , thus decreasing edema . the results showed that 2 months after surgery , retinal thickness decreased and in some cases va improved , but this improvement was not statistically significant . the decrease of macular thickness observed in our study agrees with nearly all previous studies,8,2224 but the statistically insignificant improvement in bcva is in contrast with all but one recent study,24 and one in the older literature.25 mandelcorn found that intraretinal hemorrhage and retinal thickness diminished within an average of 39 days following vitrectomy and ilm peeling.22 in this study , therefore , the last visit for evaluating the outcome of our procedure was scheduled for 2 months after surgery . in some eyes , decreased retinal thickness and improvement in me might not have been solely the result of improved physiologic features , but partly the result of inner retinal atrophy secondary to retinal ischemia . retinal atrophy might have been responsible for the apparent anatomic improvement on oct and may account for the lack of a statistically significant corresponding improvement in bcva . even when me resolves , va recovery may be limited by photoreceptor layer damage . ota and associates retrospectively examined eyes with poor va despite resolved me after brvo and demonstrated that damage to the photoreceptor layer of the retina ( as defined by lack of visualization of the related high - reflectance band on high - resolution oct ) correlated with poorer final va.26 a relatively small number of nonrandomized consecutive cases were examined in this study , and without a control group this improved anatomic outcome can not be compared with the natural history of the disease except through comparisons with published data . in the mandelcorn22 and liang et al23 studies , and in the present study , there was no difference in outcome between ischemic and nonischemic cases . ischemia was evaluated with ffa , but according to hayreh,27 this method of defining ischemia has been criticized because it provides a reliable evaluation of retinal capillary perfusion in only approximately 50%60% of cases . combined information from the assessment of relative afferent pupillary defects and electroretinography has been proposed as a superior method of evaluation that is able to differentiate ischemic from nonischemic crvo in 97% of cases . eyes in the present study , classified as nonischemic by ffa criteria , actually may have had more severe disease , and more cases of ischemic rvo might have been detected if they had been examined using alternative methods . one of the pitfalls in the present study , in comparison with mandelcorn and nrusimhadevara,8 was that in some of the authors surgeries , ilm peeling was not as complete as theirs . they peeled the ilm beyond the arcades , but in two cases in this study , the authors peeled the ilm only at the macula , and it seems that was insufficient for the drainage of fluid out of the retina ( cases 1 and 6 ) . another issue of concern is the retinal toxicity of icg.28 although using a chromovitrectomy technique for ilm peeling is inevitable , clear - cut safety profiles for the different dyes used in chromovitrectomy have not yet been established.29 finally , in this study , the duration of symptoms for all cases was more than 3 months . whether early intervention is advantageous to the final outcome was not able to be evaluated in this study . according to this and previous studies , pars plana vitrectomy with ilm peeling can be one method for decreasing me due to rvo but , because of the multiple possible causes for decreased va in rvo ( such as photoreceptor ischemia and atrophy ) , it is suggested that a larger randomized controlled trial with precise case selection using appropriate imaging and electrophysiologic techniques be undertaken . surgery should also be performed as soon as possible after diagnosis , before irreversible photoreceptor damage occurs .

highlightsthis case reports the first case to our knowledge of pathological fracture of the coronoid process of the mandible secondary to long term use of alendronic acid.demonstrates the unpredictable nature of symptoms associated with medication related osteonecrosis and its management within the hospital environment.demonstrates the rapidly progressing and unpredictable nature of the spread of the necrotic process in medication related osteonecrosis.clear clinical photographs of the surgical procedure involved in the removal of necrotic bone and curettage of the surgical site in medication related osteonecrosis .

a 77-year old female was referred to our clinic with a prolonged period of delayed healing following the uneventful extraction of the lower right second molar tooth in may 2007 . at this point the patient had been taking alendronic acid 70 mg orally once per week for seven years . clinical examination revealed only mild erythema overlying the socket , with no evidence of any osteonecrotic process , either clinically of radiographically . the patient attended the following year in november 2008 with right sided facial pain following recent curettage of the affected socket by her dentist . the patient was discharged following an extended course of penicillin v and metronidazole accompanied with chlorhexidine mouthwashes . over three years later in june 2012 the patient re - attended with a repeat occurrence of her symptoms . clinically there was no evidence of infection or discharge but radiographically there remained a clearly demarcated radiolucency over the right posterior mandible ( fig . the histopathology findings confirmed bone necrosis consistent with mronj , and ruled out dysplasia or malignancy . the patient s most recent attendance was in october 2013 where she attended with increasing trismus and some swelling over the right posterior mandible . radiographic examination revealed a much more extensive area of necrosis at the right body and ramus of the mandible spreading to involve the coronoid process and the appearance of a pathological fracture ( fig . 2 ) . a ct image was taken to assess the extent of the necrosis . exploration of the right side of the mandible with removal of the fractured coronoid process and debridement of necrotic bone was carried out at the royal surrey county hospital in december 2013 . an incision was made to explore the body , ascending ramus , and coronoid process of the mandible . the coronoid process was gripped firmly to prevent the temporalis pulling the fragment superiorly , and gently removed ( fig . once the coronoid process was removed , gentle curettage of the necrotic bone was carried out using a slow hand piece and saline irrigation . necrotic bone was removed leaving a margin of bleeding bone with normal appearance thus indicating sufficient metabolic potential for healing to take place ( fig . 8) [ 36 ] . the patient was admitted for two nights following the procedure where she received intravenous metronidazole , and was discharged with oral antibiotics and chlorhexidine mouthwashes four times per day for two weeks . this case demonstrates the unpredictable nature of mronj and how the disease can progress to cause significant morbidity . in this case extensive surgery was required to remove the necrotic fragments of bone with no guarantee that the necrosis will stop spreading . at present , the incidence of mronj in patients taking alendronic acid for osteoporosis is 1:10001:1,700 however , this incidence increases with the duration of treatment [ 79 ] . our case illustrates the extent of the damage that can be caused when straightforward dental procedures are carried out in low risk patients taking oral bisphosphonates . it is important to realise that osteonecrosis may remain asymptomatic for long periods in the absence of infection . it seems a matter of great importance that national evidence based guidelines are published on the dental treatment of patients taking bisphosphonates , and that further information regarding the possible consequences of bisphosphonate use is given to general medical practitioners . there are no conflicts of interest . written informed consent for publication in print and electronic form , and publication of radiographic and photographic images written informed consent for publication in print and electronic form , and publication of radiographic and photographic images was granted by the patient . authorsconception and design of study / review / case seriesacquisition of data : laboratory or clinical/ literature searchanalysis and interpretation of data collecteddrafting of article and/or critical revisionfinal approval and guarantor of manuscriptadam jowett clinical/ literature searchanwer abdullakutty clinical/ literature searchmalcolm bailey clinical/ literature search

we screened 768 tick pools containing 6,962 ticks from khammouan province , laos , by using quantitative real - time pcr and identified rickettsia spp . , ehrlichia spp . , and borrelia spp . sequencing of rickettsia spp.positive and borrelia spp.positive pools provided evidence for distinct genotypes . our results identified bacteria with human disease potential in ticks in laos .

we collected ticks in nakai district , khammouan province , during the dry seasons ( december april ) during 20122014 , as previously described ( 4 ) ( technical appendix figures 1 , 2 ) . a total of 6,692 ticks were pooled ( n = 768 pools , 110 ticks / pool ) according to genus , sex , developmental stage , collection period , and site . we extracted dna by using the nucleospin 8 virus extraction kit ( macherey - nagel , dren , germany ) . pools were screened by using single quantitative real - time pcrs specific for rickettsia spp . five microliters of diluted ( 1:10 ) template containing 1 platinum supermix - udg ( invitrogen , carlsbad , ca , usa ) and bovine serum albumin ( 40 mg / ml ) were used for each assay . results were considered positive if they had a cycle quantitation ( cq ) value < 40 and likely positive if they had a cq value 4045 ( 9 ) . sequencing was attempted for pools with cq values < 40 ( online technical appendix table 2 ) and performed by macrogen ( seoul , south korea ) . consensus sequences were analyzed by using clc main workbench 7 ( http://www.clcbio.com/products/clc-main-workbench/ ) and blast ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) and submitted to genbank . phylogenetic trees were constructed by using the kimura-2 parameter model and the neighbor - joining method . , 36.3% ( 279/768 ) a. testudinarium , and 3.8% ( 29/768 ) dermacentor auratus . of the pools , 3% ( 23/768 ) contained adults , 36.5% ( 280/768 ) contained larvae , and 60.5% ( 465/768 ) contained nymphs ( table 1 ) . * were identified in 5.7% ( 44/768 ) of pools , and an additional 2.3% ( 18/768 ) of pools were likely positive for rickettsia spp . sequences consistent with 5 described rickettsia species or genotypes were identified : r. tamurae , r. japonica , rickettsia sp . the tree was constructed by using partial nucleotide sequences ( 350 bp ) of the 17-kda gene , the kimura 2-parameter model , and the neighbor - joining method . candidatus rickettsia laoensis ( pool 447 ) was identified in 1 haemaphysalis sp . pool . phylogenetic analysis of 28452920-bp concatenated sequences of glta , sca4 , and ompb genes suggested that this bacteria belonged to the r. massiliae group of rickettsiae ( technical appendix figure 3 ) . candidatus rickettsia mahosotii ( pools 81 and 372 ) was identified in haemaphysalis spp . and a. testudinarium pools . phylogenetic analysis of glta , sca4 , and ompb genes suggested that this bacteria belonged to the r. rickettsii group ( technical appendix figure 3 ) . phylogenetic analysis of glta , 17-kda , and ompb genes suggested a relationship with the r. helvetica group ( technical appendix figure 4 ) . in addition , 15 a. testudinarium pools showed dual peaks for 17-kda gene sequences , which suggested the presence of r. tamurae and rickettsia sp . sequencing of sca4 , ompa , and ompb genes from 1 of these pools ( pool 239 ) identified unique sequences ( table 2 ; technical appendix figure 4 ) . b897888 ) and belong to the relapsing fever group of borrelia ( figure 2 ) . the tree was constructed by using partial nucleotide sequences ( 299323 bp ) of the flab gene , the kimura 2-parameter model , and the neighbor - joining method . ( table 1 ) ; an additional 6 pools ( 0.8% ) were likely positive . nymph pool ( pool 357 ) was obtained , and this sequence showed 100% identity ( 116/116 bases ) with the genus ehrlichia . no pools were positive for anaplasma spp . , but 2 were likely positive ( table 1 ) . ( n = 511 ) , 1 pool ( 0.2% ) was positive , and 4 pools were likely positive for c. burnetti . the 1 tick that contained a blood meal ( a. testudinarium nymph ) showed negative results by screening pcrs . infections with r. tamurae ( 2 ) and r. japonica are well described in southeast asia ( 10 ) . however , the pathogenicity of rickettsia sp . twkm01 ( 11 ) , rickettsia sp . kagoshima6 genotypes ( 13 ) and potential novel candidatus rickettsia laoensis , candidatus rickettsia mahosotii , and candidatus rickettsia khammouanensis is unknown . candidatus rickettsia khammouanensis is phylogenetically related to r. helvetica , for which there is serologic evidence for its role as a human pathogen in laos ( 2 ) . unique ompa , ompb , and sca4 sequences identified in this study ( table 2 ) might indicate the presence of rickettsia sp . att ( 12 ) , which was previously believed to be identical to r. tamurae ( 14 ) , and suggests that it might be a distinct species . further studies , including whole - genome sequencing , are required to identify and confirm these novel genotypes and understand their role in human disease . pools were shown to have high concordance with the shiretoko haemaphysalis borrelia isolated from haemaphysalis spp . ticks and deer in japan ( 15 ) . the species belongs to the relapsing fever group of borrelia and is related to b. lonestari . sequence data for ehrlichia spp . indicated the presence of these bacteria but were not sufficient to identify them to the species level . the cq values were high ( 4045 ) for anaplasma spp . , but no sequence data were obtained . were screened by using primers for is1111 , which are not specific for c. burnetii , and no confirmatory sequence data were obtained . because of limited reagents , screening of all 768 pools for coxiella spp . was not completed . second , sequences obtained from some a. testudinarium pools had dual peaks , suggestive of multiple infections , and could therefore not be interpreted . third , ticks were collected only from 1 area in laos ( khammouan province ) ; thus , extrapolating findings to the entire country must be done cautiously . these findings emphasize the need for further research of tick - associated bacteria and their role in human disease . additional information on large - scale survey for tickborne bacteria , khammouan province , laos .

alzheimer 's disease ( ad ) is a neurodegenerative brain disorder associated with the loss of synapses between neurons in the brain . synaptic cell adhesion molecules are cell surface glycoproteins which are expressed at the synaptic plasma membranes of neurons . these proteins play key roles in formation and maintenance of synapses and regulation of synaptic plasticity . genetic studies and biochemical analysis of the human brain tissue , cerebrospinal fluid , and sera from ad patients indicate that levels and function of synaptic cell adhesion molecules are affected in ad . synaptic cell adhesion molecules interact with a , a peptide accumulating in ad brains , which affects their expression and synaptic localization . synaptic cell adhesion molecules also regulate the production of a via interaction with the key enzymes involved in a formation . a-dependent changes in synaptic adhesion affect the function and integrity of synapses suggesting that alterations in synaptic adhesion play key roles in the disruption of neuronal networks in ad .

cell adhesion molecules ( cams ) are cell surface glycoproteins located at the cell surface plasma membrane of neurons and other cells . cams have a large extracellular domain and are either transmembrane proteins or attached to the plasma membrane via a glycosylphosphatidylinositol ( gpi ) anchor . the extracellular domains of cams mediate cell adhesion by either forming homophilic adhesion bonds via binding to the same molecules on cell surface membranes of adjacent cells or interacting heterophilically with other proteins on the cell surface membranes of adjacent cells or in the extracellular matrix . cams accumulating at synapses between neurons are often called synaptic cams and represent members of the major families of cell adhesion molecules , including immunoglobulin superfamily ( igsf ) cams , cadherins , integrins , neuroligins , and neurexins , and also other cell surface proteins , which mediate cell adhesion , such as cellular prion protein ( prp ) and amyloid precursor protein ( app ) ( figure 1 ) . synaptic cams perform numerous functions at synapses ( figure 1 ) . in developing neurons , cams promote mechanical stabilization of the contacts between axons and dendrites of neurons and formation of synapses . synaptic cams also play key roles in the establishment of neurotransmission by recruiting other synaptic components , such as synaptic scaffolding proteins , which interact with the intracellular domains of synaptic cams , and associated neurotransmitter receptors ( figure 1 ) , and by inducing the maturation of the neurotransmitter release machinery . in mature neurons , cams play a role in the stabilization of the synapse ultrastructure [ 57 ] , regulation of the neurotransmitter release [ 8 , 9 ] , and synaptic remodeling and plasticity [ 1013 ] . the multiple roles of synaptic cams in regulation of synapse formation and function have been described in a number of recent reviews [ 14 , 15 ] and are discussed here mostly in the context of alzheimer 's disease . alzheimer 's disease ( ad ) is a neurodegenerative brain disorder , which predominantly affects the aging population . synapse loss in ad has been linked at least partly to the toxicity induced by a , a peptide that accumulates in the brains of ad patients [ 1719 ] . synaptic cell adhesion is directly involved in ad pathogenesis , since app is a precursor protein of the a peptide and also a synaptic cell adhesion molecule playing a role in regulation of synaptic morphology , synaptic plasticity , and hippocampus - dependent behavior . functions of app in synapses and molecular mechanisms of a formation are the subject of a number of recent reviews [ 2123 ] and are not discussed here . in this review , we summarize current data on the changes in the levels and function of other synaptic cams in ad brains and their complex interactions with a suggesting that abnormal function in different synaptic cams can be an important factor contributing to synapse dysfunction in ad . the involvement of cams in ad is suggested by genome - wide association studies ( gwas ) . significantly altered expression of cam pathway genes in ad was found in the samples from the cerebellum and temporal cortex of ad - affected individuals and ad - nonaffected controls . besides app , among synaptic cams found to be associated with the risk of ad , prnp gene coding for prp has been identified as an ad susceptibility gene by systematic meta - analysis of ad genetic association studies . the methionine / valine ( m / v ) polymorphism at codon 129 within the prnp gene , which represents a known risk factor for creutzfeldt - jakob disease ( cjd ) , has also been reported to be a risk factor for early onset ad [ 2628 ] . single nucleotide polymorphisms ( snps ) in the neural cell adhesion molecule 2 ( ncam2 ) , a synaptic igsf cam highly expressed in hippocampal synapses , have been reported as a risk factor related to the progression of ad in the japanese population . snps in the ncam2 gene also show association with levels of a in the cerebrospinal fluid in humans , suggesting that ncam2 is involved in the pathogenic pathway to the senile plaques that concentrate in ad brains . in another large gwas involving over 16,000 individuals , snps in contactin-5 , another member of the synaptic igsf cams localizing to the presynaptic membranes , were shown to be significantly associated with ad . the junctional adhesion molecule 2 ( jam2 ) is another member of igsf potentially linked to ad . jam2 is localized to tight junctions in epithelial and endothelial cells but is also expressed in retinal ganglion cells . the link between jam2 and ad is also suggested by a study reporting chromosomal 21 region duplication spanning 0.59 mb and comprising jam2 , app , and some other genes in a patient with ad . whether jam2 functions in the regulation of synapses in neurons is , however , not known . the association with ad was also observed for snps in the gene coding for the leucine - rich repeat transmembrane neuronal 3 ( lrrtm3 ) synaptic cam , which is highly expressed in the hippocampus . meta - analysis of five gwas also identified the gene coding for neurexin-3 as a gene playing a role in susceptibility to ad in males . changes in the levels of synaptic cams in ad brains have been reported in a number of studies performed over the last 25 years . reduced levels of the largest ncam isoform with the longest intracellular domain , ncam180 , but not total ncam levels have been reported in one of the early studies comparing samples from control and ad frontal cortex by quantitative crossed immunoelectrophoresis suggesting changes in the expression of ncam in ad . in later studies , analysis of control and ad brain sections by immunohistochemistry with antibodies against ncam found significantly fewer ncam positive neurons in the frontal cortex of ad - affected individuals when compared to normal aging individuals . in agreement , the levels of ncam were shown to be reduced in frontal and temporal cortex from ad patients by elisa . interestingly , there was little difference in the levels of ncam in the occipital cortex and hippocampus of control and ad patients [ 39 , 41 ] . however , immunohistochemical analysis of the ad hippocampus using antibodies against polysialic acid ( psa ) , a unique carbohydrate attached predominantly to ncam , revealed an increase in the immunoreactivity and numbers of psa - ncam positive neurons in ad hippocampus and especially in the dentate gyrus indicating changes in the posttranslational processing of ncam . psa - ncam is highly expressed in the developing nervous system , but its expression in the mature nervous system is restricted to brain areas undergoing plastic changes , suggesting that an increase in psa - ncam in ad is related to extensive neuronal remodeling in ad brains . levels of contactin-2 , a gpi anchored igsf cam also called transient axonal glycoprotein 1 ( tag-1 ) , were shown by western blot to be reduced in the temporal lobe of ad patients . contactin-2 is cleaved by -site amyloid precursor protein - cleaving enzyme 1 ( bace1 ) and its levels in ad brains inversely correlate with bace1 levels and amyloid plaque density suggesting that an increase in bace1 activity observed in late - onset ad [ 4549 ] results in increased contactin-2 cleavage . bace-1 also cleaves other synaptic cams , such as igsf cam l1 and the close homologue of l1 ( chl1 ) [ 50 , 51 ] . the intracellular domain of l1 is also cleaved by -secretase in human carcinoma cells , and -secretase induced proteolytic cleavage of l1 is increased in a mouse model of ad , which carries human app with the pathogenic swedish mutation and the l166p mutated human presenilin-1 . changes in the activity of bace-1 and -secretase may therefore affect the expression of a number of other synaptic cams in ad brains . in addition to igsf cams , levels of prp analyzed by western blot were also found to be decreased in the hippocampus of patients with sporadic ad but not with familial ad . levels of prp are also lower in the temporal cortex samples of ad patients [ 54 , 55 ] . levels of n - cadherin are also reduced in the temporal cortex of ad patients . in contrast , western blot analyses have not revealed significant changes in the levels of contactin-5 in the temporal cortex of ad patients and levels of full length n - cadherin in the superior frontal gyrus of ad patients . levels of platelet endothelial cell adhesion molecule 1 ( pecam1 ) , an igsf cam , were also similar in frontal and temporal cortex of control subjects and moderate to severe ad patients . therefore , expression of only a subset of synaptic cams appears to be affected in ad and only in some brain regions . interestingly , in a recent study , levels of ncam2 were shown by western blot to be increased in the hippocampus of ad patients but strongly reduced in synaptosomes isolated from this brain region ( figure 2 ) . levels of ncam2 were not significantly affected in the temporal cortex and cerebellum of ad patients . these observations indicate that changes in the total protein levels or the lack of such changes does not necessarily correlate with the changes in the subcellular localization and function of synaptic cams . changes in the levels of other synaptic cams at synapses in ad brains and whether alterations in the overall levels of other synaptic cams reflect changes in their synaptic localization remain to be investigated in the future studies . in addition to changes in the levels of the full length synaptic cams , changes in the levels of the proteolytic products of synaptic cams have also been found in ad brains . interestingly , changes in the proteolytic products of synaptic cams do not necessarily correlate with the changes in the total protein levels . while the total levels of n - cadherin appear to be unaffected in the superior frontal gyrus of ad patients , the levels of ectodomain - shed c - terminal fragment of n - cadherin the levels of the extracellular domains of ncam2 proteolytically released from the neuronal cell surface are increased in ad hippocampus ( figure 2 ) . this increase in the levels of proteolytic products of ncam2 inversely correlates with the levels of full length ncam2 at synapses , while the total levels of ncam2 are also increased in ad hippocampus ( figure 2 ) . it is therefore possible that changes in the proteolytic products of synaptic cams in ad brains reflect changes in their proteolysis at specific subcellular locations , such as synapses , rather than changes in the overall turnover of these proteins . a number of studies indicate that the proteolytic products of cams are also present at varying levels in the cerebrospinal fluid ( csf ) and serum of humans . western blot analyses with antibodies specific to different portions of these molecules show that these proteolytic products are detectable with the antibodies against the epitopes within their extracellular domains while the intracellular domains are not detectable . these observations indicate that the proteolytic products of cams in csf and serum represent fragments of the extracellular domains of cams possibly released to csf by shedding from the cell surface of neurons in the brain . proteolytic products of several cams have been reported to be increased in csf and serum of ad patients . for example , csf levels of l1 analyzed by elisa have been reported to be significantly increased in ad . this study also reported an increase in the csf levels of ncam , which , however , was not statistically significant when compared to normal controls . increased levels of several proteolytic products of ncam were also found in the sera of ad patients . in contrast , elisa analysis has not revealed significant differences in the levels of neuronal cell adhesion molecule ( nrcam ) , l1 family member , in csf samples from healthy controls and ad patients . analysis of the levels of the proteolytic products of cams has therefore been proposed to be useful in diagnostics of ad . it should be noted , however , that levels of the proteolytic products of such cams as ncam or l1 in csf and serum samples of healthy individuals and ad patients overlap considerably [ 60 , 61 ] . also , changes in csf levels of these products are often not specific to ad . for example , levels of the proteolytic products of l1 are also increased in vascular dementia and dementia of mixed type . levels of the proteolytic products of ncam are increased in csf of people suffering from schizophrenia and bipolar mood disorder type i or recurrent unipolar major depression , but not in bipolar mood disorder type ii patients . however , levels of ncam are not changed in the serum of patients with autism , although levels of ncam180 protein but not mrna are reduced in the brains of these patients . also , in contrast to ad , csf levels of l1 are decreased in schizophrenia . therefore , analysis of specific isoforms and cleavage products derived via different proteolysis pathways might be required to establish proteolytic products of cams as markers of specific neurologic conditions including ad . a number of observations indicate that synaptic cams act as receptors for a oligomers at the synaptic sites . the extracellular domain of l1 but not the extracellular domain of chl1 interacts with a in a label - free binding assay ( figure 3 ) . the fibronectin type iii homologous repeats 13 of the extracellular domain of l1 mediate this effect . interestingly , the recombinant extracellular domain of l1 , but not the recombinant extracellular domain of chl1 , inhibits aggregation of a in vitro . furthermore , overexpression of l1 by injection of adenoassociated virus encoding l1 decreases the a plaque load , levels of a42 , a42/40 ratio , and astrogliosis in a mouse model of ad , which carries human app with the pathogenic swedish mutation and the l166p mutated human presenilin-1 . the extracellular domain of ncam2 also binds to a oligomers both in vitro and in vivo in cultured mouse hippocampal neurons and in the hippocampus of a generating transgenic mice overexpressing human app containing the pathogenic swedish mutation . a oligomers also directly associate with the n - terminus of prp both in vitro and in the human ad brain , with the binding sites located within residues 2327 and 95110 of prp [ 6771 ] . interaction of prp with a is a function of a load in the brain and does not depend on prp levels . prp interacts with other synaptic proteins , including n - methyl - d - aspartic acid- ( nmda- ) type glutamate receptors and ncam , and binding of a oligomers to prp can interrupt the physiological interactions of prp at synapses , resulting in disturbed neuronal communication . prp deficient mice are resistant to the neurotoxic effect of a oligomers , and antibodies against prp or prp peptides prevent a oligomer - induced neurotoxicity indicating that prp is involved in the molecular pathways activated by a oligomers to induce neuronal cell death . the cleavage fragment of prp containing binding sites for a strongly suppresses a toxicity in cultured mouse hippocampal neurons and in vivo in mice after intracerebroventricular injections of a [ 69 , 76 ] . however , memory impairment induced by injection of a oligomers is not reduced in prp knockout mice , ablation or overexpression of prp has no effect on the impairment of hippocampal synaptic plasticity in a transgenic model of ad , and synaptic depression , reduction in spine density , or blockade of ltp is induced by a in organotypic hippocampal slice neurons from both wild type and prp knockout mice . neuroligin-1 is enriched in excitatory synapses and its extracellular domain binds to a in vitro and in cultured rat hippocampal neurons and rat cerebral cortex [ 80 , 81 ] . neuroligin-1 acts as a nucleating factor during the a aggregation process , stimulating the formation of a oligomers . the soluble extracellular /-hydrolase - fold ( che - like ) domain of neuroligin-1 reduces the a-induced reduction in synaptic density in cultured rat hippocampal neurons and in field excitatory postsynaptic potentials ( fepsp ) in rat hippocampal slices possibly by competing with the synaptic neuroligin-1 for binding to a . indirect observations also suggest that a interacts with integrins since a toxicity was inhibited in human neurons pretreated with adhesion - blocking antibodies against different subunits of integrins , and in particular 1 , 2 , and v . inhibition of 11 integrin has also been shown to reduce a toxicity in rat hippocampal cultures . a toxicity was also inhibited by disintegrin echistatin , a peptide isolated from snake venom that has been shown to inhibit rgd - dependent integrins such as v1 and by integrin ligands such as vitronectin , fibronectin , and superfibronectin suggesting that integrin ligands compete with a for binding to integrins . application of a also reduces the overall expression of n - cadherin in cultured mouse cortical neurons suggesting that a can bind to n - cadherins , although a reduction in n - cadherin proteolysis after application of a has been reported in another study . the association of n - cadherin with app in mouse brains has been shown by coimmunoprecipitation experiments . in an unbiased search for the binding partners of app using time - controlled transcardiac perfusion cross - linking followed by high stringency immunoaffinity purification and tandem mass spectrometry , several other cell adhesion molecules were identified including prp and igsf cams thy-1 , contactin , ncam1 , and neurofascin . in spite of homology to ncam2 , ncam1 binds to a region of app which is different to the a-containing region indicating that these interactions may play a role in physiological functions of both molecules . in agreement binding of contactin-2 to app increases the release of the intracellular domain of app through -secretase - dependent cleavage . contactin-2 competitively inhibits the binding of app to transforming growth factor 2 ( tgf2 ) . binding of tgf2 to app induces neuronal cell death and this effect is inhibited by tag-1 suggesting that tag-1 regulates interactions of app with extracellular ligands . bace1 is a potential therapeutic target for ad since bace1 cleavage of app is the rate limiting step in a production . synaptic cell adhesion molecules have been shown to play a role in regulation of bace1 activity . in a high - throughput sirna screen assessing 15,200 genes for their role in a secretion , lrrtm3 has been identified as a neuronal gene that promotes app processing by bace1 . knockdown of lrrtm3 expression using sirna results in reduced secretion of a in cultured cells and primary neurons , while overexpression of lrrtm3 increases a secretion suggesting that lrrtm3 promotes bace1 activity . in contrast , overexpression of prp results in inhibited bace1-mediated cleavage of app and reduced a production , while a production is increased in the brains of prp knockout mice and in cultured n2a cells after sirna mediated knockdown of prp expression suggesting that prp inhibits bace1 activity . in agreement , in a follow - up study , prp has been shown to interact with the prodomain of bace1 in the trans - golgi network and regulate targeting of bace1 to the cell surface and endosomes where it preferentially cleaves app . prp reduces bace1-mediated cleavage of wild type app , but not human app with the swedish and indiana familial mutations , suggesting that prp may play a role in sporadic ad but not in familial ad . interestingly , the region at the extreme n - terminus of prp , which is critical for the interaction or prp with bace-1 and prp - dependent inhibition of app - cleaving activity , also contains the binding site for a oligomers . these observations suggest that prp can play a protective role ( inhibition of bace1 ) and pathogenic role ( binding of toxic a oligomers ) in ad and also suggest that the protective function of prp can be affected by a oligomers . an increase in a secretion is also observed in cells cotransfected with n - cadherin [ 85 , 95 ] . n - cadherin promotes cell surface expression of -secretase and increases accessibility of -secretase to app . altogether , these observations thus indicate that synaptic cams are involved in regulation of the key enzymes involved in a production . inhibition of n - cadherin function by blocking inp peptides , which mimic a short sequence in the ec1 domain of n - cadherin and thus impair the homophilic transsynaptic interaction of n - cadherin molecules , accelerates the a-induced synapse impairment characterized by a reduction in the frequency of the ampa receptor - mediated miniature excitatory postsynaptic currents ( ampa mepscs ) and reduced density of synaptic boutons along dendrites in cultured cortical neurons . similar effects are observed when n - cadherin function is inhibited by expression of the dominant - negative , truncated n - cadherin lacking the extracellular cadherin domains , or by overexpression of the ectodomain - shed c - terminal fragment of human n - cadherin , which accumulates in ad brains . it is noteworthy that the ectodomain - shed c - terminal fragment of human n - cadherin is further cleaved by -secretase , and inhibition of -secretase activity also accelerates the a-induced synapse impairment . inhibition of n - cadherin function alone has no effect on the numbers of synapses and frequency of ampa mepscs . interestingly , disruption of ncam2-mediated synaptic adhesion using recombinant extracellular domains of ncam2 ( ncam2-ed ) results in a reduction in synapse density along dendrites of hippocampal neurons and dispersion of ampa receptors from synapses . ncam2-ed accumulates in ad hippocampus and its effect on the synapse integrity is similar to and not additive with the effect of a . it is therefore possible that a-dependent proteolysis of ncam2 is one of the initial synapse - destabilizing effects of a , which is then followed by disruption of n - cadherin containing adhesion complexes resulting in the complete synapse disassembly . the complex formed by a and neuroligin-1 also contains glun2b but not glun2a subunits of nmda receptors suggesting that a can directly affect the function of neuroligin-1 in anchoring nmda receptors at synapses . whether binding of a to other synaptic cams directly contributes to the synapse loss has to be investigated in the future studies . while a number of observations indicate that synaptic cell adhesion molecules are affected in ad , our understanding of the molecular and cellular mechanisms underlying these changes and their role in the disease progression is still very incomplete . further studies assessing levels of synaptic cams specifically at synapses are needed to understand whether changes in the overall levels of these cams reflect changes in the synaptic adhesion . whether an increase in the levels of specific proteolytic products of cams in csf and sera of ad patients reflect the a-dependent proteolysis of cams at synapses is an interesting possibility which can be analyzed in the future studies . since synaptic cams play key roles in the maintenance of synapse integrity and function by interacting with synaptic scaffolding proteins and neurotransmitter receptors , further analysis of the effects of a-dependent disruption of synaptic adhesion at the synaptic level may help to understand the molecular mechanisms of the initial stages of ad . furthermore , a number of reports showing that the a toxicity can be reduced by targeting synaptic cams indicate that synaptic cams deserve further consideration as molecular targets in designing new treatments of ad .

nucleic acids of human papillomavirus ( hpv ) isolated by manual extraction method ( amplilute ) and automated magna pure system were compared and evaluated with cytohistological findings in 253 women . the concordance level between amplilute and magna was very good 93.3% ( = 0.864 , p < .0001 ) . overall hpvpositivity detected by amplilute was 57.3% ( 30.4% as single and 27% as multiple infections ) in contrast to magna 54.5% ( 32% and 23% , resp . ) . discrepant results observed in 25 cases : 11 magna()/amplilute(+ ) , 10 of which had positive histology ; 5 magna(+)/amplilute( ) with negative histology ; 8 magna(+)/amplilute(+ ) : in 7 of which amplilute detected extra hpv genotypes and 1 magna(invalid)/amplilute(+ ) with positive histology . both methods performed well when compared against cytological ( area under curve ( auc ) of amplilute 0.712 versus 0.672 of magna ) and histological diagnoses ( auc of amplilute 0.935 versus 0.877 of magna ) , with amplilute showing a slightly predominance over magna . however , higher sensitivities , specificities , and positive / negative predictive values were obtained by amplilute .

it is now well established and widely accepted that virtually all cervical cancer and its immediate precancerous lesions arise from persisting cervical infection by some highly oncogenic hpv genotypes [ 1 , 2 ] . the most important of these hpv genotypes are hpv16 and hpv18 which account for ~70% of all invasive cervical cancers with minor variations in this percentage between continents . fifteen hpv genotypes have been to date classified as high - risk ( hr ) types ( 16 , 18 , 31 , 33 , 35 , 39 , 45 , 51 , 52 , 56 , 58 , 59 , 68 , 73 , and 82 ) , 3 as probably hr ( 26 , 53 , 66 ) and 12 as low - risk ( lr ) ( 6 , 11 , 40 , 42 , 43 , 44 , 54 , 61 , 70 , 72 , 81 , and cp6108 ) [ 1 , 4 ] . the majority of hpv infections are transient , but persistence of an hr hpv is a significant risk factor for the development of cervical cancer . it could be a genetic predisposition with an inadequate immune response and/or possible uncontrolled reaction with tumor suppressor genes [ 5 , 6 ] . type - specific detection of hpv is increasingly important for monitoring the impact of hpv vaccine implementation and as a tool for cervical cancer screening . as a consequence , the commercial hpv detection kits : hybrid capture ii ( digene corporation , gaithersburg , md , usa ) , cervista hpv hr ( third wave technologies , inc . , madison , usa ) and cervista 16/18 tests are approved by the fda for use in routine screening of hpv . however , the above assays are unable to discriminate specific genotypes or to identify infections involving multiple genotypes and the cervista assay detects only two hpv types ( types 16 and 18 ) . various molecular assays for hpv detection and typing have been used in epidemiological studies , and they are based on two different technologies : ( 1 ) hybridization - based assays ( e.g. , hc ii ) and ( 2 ) pcr - based tests ( e.g. , gp5+/gp6 + , pgmy09/11 , inno - lipa hpv genotyping ( innogenetics , belgium ) , linear arrays hpv test ( roche molecular systems , inc . branchburg , nj , usa ) , clart hpv2 ( genomica , madrid , spain ) . the advantages and disadvantages of these two basically different methodologies have been extensively discussed [ 719 ] . the qualitative linear array hpv ( la - hpv ) hpv genotyping test , developed by roche molecular systems offers a reliable , sensitive , and standardized approach for hpv typing in cervical specimens . it is distributed as a research use only but it has been submitted for fda review . this test utilizes amplification of target dna by pcr and nucleic acid hybridization for the detection of 37 types in cervical cells collected into an lbc media . dna extraction , pcr amplification , hybridization of the amplified products with specific probes and colourimetric detection on the hybrids on strip [ 13 , 17 , 2022 ] . current specimen processing protocols recommend the use of manual extraction of dna using the amplilute liquid media extraction kit , based on the qiaamp method ( qiagen , inc . , an alternative method for dna extraction is the automated magna pure lc extraction system , developed by the same company . the objective of this study was to evaluate and compare the automated magna pure dna extraction method with the amplilute dna extraction method in detecting hpv dna form thinprep pap tests using the linear array ( la ) hpv genotyping and detection assays and also to correlate these results to cytological and histological diagnosis . in the present study , cervical brush specimens were obtained from women aged from 17 to 70 years who attended the gynecologic outpatient clinic of attikon university hospital , athens , greece , for opportunistic examination , between july 2009 and may 2010 . women considered eligible for the study if they fulfilled the following criteria : ( a ) they agreed to undergo colposcopy and if necessary cervical biopsy and ( b ) there was enough residual biological material , after cytological examination , for the two molecular assays to be completed . this patient population does not represent the general population of women attending public screening programs . samples of thinprep pap tests were collected by means of a brun's - like brush . the preservcyt vials ( corporate headquarters : hologic , inc . , ltd . , uk ) , containing the cell samples were addressed to the department of cytopathology of the aforementioned hospital for preparation of thin - layer slides using the thinprep 2000 automated slide processor ( corporate headquarters : hologic , inc . , cytological findings were interpreted according to the bethesda classification system and were classified as follows : ( a ) within normal limits ( wnl ) , ( b ) atypical squamous cells of undetermined significance ( asc - us ) , ( c ) low - grade squamous intraepithelial lesion ( lsil ) , and ( d ) high - grade squamous intraepithelial lesion ( hsil ) . the cytopathologists and the biologist conducting hpv testing were all blinded to the clinical profile to ensure unbiased reporting . the histological evaluation revealed the following categories : negative hpv , cin 1 , cin 2 , and cin 3 . in case histology showed a cin 2 or cin 3 , the patient was referred for appropriate treatment . after slide preparation for cytological examination , the remaining preservcyt samples were vortexed vigorously for 15 sec to maximize homogeneity and two aliquots of 250 l and 1 ml were generated from each clinical specimen . dna was isolated using two different procedures ( i ) a 250 l aliquot was extracted by amplilute liquid media kit ( roche molecular systems ) in conjunction with a qiavac 24 plus vacuum system , according to the manufacturer 's instructions in the product insert and ( ii ) a 1 ml aliquot was extracted by magna pure lc extraction system using the dna - i kit ( blood cells high - performance protocol ) ( roche molecular systems ) . briefly , for the manual extraction , samples and hpv positive / negative controls were processed in parallel . clinical samples were mixed by vortexing to form a homologous state , and 250 l were removed and lysed with proteinase k solution and buffer atl at 56c for 30 min . the samples underwent a second incubation at 70c for 15 min in the presence of buffer al containing a carrier rna . the lysate was then transferred to vacuum columns where isolation and purification of dna was completed via washing of different solutions to bind dna and remove other cellular materials . specimens and controls were immediately stored at 2c8c for up to 7 days or frozen at 20c for up to 8 weeks . for automated extraction , the samples were prepared using a modified procedure involving the centrifugation of 1 ml aliquots of the preservcyt samples at 13000 g for 20 min prior to discarding of the supernatant . the resulted cell pellets were resuspended into 200 l of sterile phosphate - buffered saline , and the procedure of automated extraction was followed according to the manufacturer 's instruction , using the dna - i kit . the method is based on magnetic - bead technology with a special buffer containing chaotropic salts and proteinase k. nucleic acids are bound to the surface of the magnetic glass particles . cellular debris was removed by several washing steps , and the purified nucleic acids were eluted . 100 l in volume of extracted genomic dna product was obtained , after the magnetic beads were separated from the solution . specimens and controls were immediately stored at 2c8c for up to 7 days or frozen at 20c for up to 8 weeks . after nucleic acid purification all samples were analyzed by la hpv assay for hpv genotyping . the la genotyping test use a pool of biotinylated primers designed to amplify an approximately 450 bp sequence within the polymorphic l1 region of the genome of the 37 hpv genotypes . an additional 268 bp primer pair which targets the human -globin gene is included in the assay to provide a control for cell adequacy , extraction , and amplification . pcr was carried out on each of the samples and controls , using the linear array hpv genotyping mastermix which contains : tris buffer , potassium chloride , amplitaq , gold dna polymerase ( microbial ) , amperase , ( uracil - n - glycosylate ) enzyme ( microbial ) , datp , dctp , dutp , dgtp , dttp , each of upstream and downstream primers ( biotinylated ) and -globin primers , sodium azide , magnesium chloride , and amaranth dye . the reaction mixture contained 50 l of hpv mastermix and 50 l of eluted dna . the amplification was performed on the applied biosystems gold - plated 96-well geneamp pcr system 9700 ( applied biosystems , foster city , calif , usa ) using the following thermal profile : 2 min at 50c , 9 min at 95c ; 30 sec at 95c , 1 min at 55c , 1 min at 72c ( 40 cycles ) with a ramp rate set at 50% followed by 5 min at 72c and a final hold at 72c indefinitely . pcr amplicons were immediately denatured by the addition of 100 l of ( dn ) denaturation reagent and stored at 4c for further analysis within 7 days . the detection of the hpv genotypes was carried out using the la hpv detection kit . once the amplification was completed , 75 l of denatured amplicon were added to the linear array strips that contain multiple copies of hpv genotype - specific probes in a defined area for all 37 genotypes and the -globin reference lines . the hpv types detected are hpv6 , 11 , 16 , 18 , 26 , 31 , 33 , 35 , 39 , 40 , 42 , 45 , 5156 , 58 , 59 , 61 , 62 , 64 , 6673 , 8184 , is39 , and cp6108 . the biotin - labeled amplicon was bound to the strips using a hybridization buffer in a shaking waterbath at 53c . once bound , the strips were washed at high stringency to remove nonbound material and streptavidin - horseradish peroxidase conjugate was then added and bound to the biotin - labeled amplicon hybridized to the oligonucleotide probes . the strips were then washed with a substrate solution containing hydrogen peroxide and 3,3,5,5-tetramethylbenzidine ( tmb ) . in the presence of hydrogen peroxide , the bound streptavidin - horseradish peroxidase catalyzed the oxidation of tmb to form a blue - colored complex , which precipitated at the probe positions where hybridization had occurred ( colourimetric determination ) . this color precipitation allowed for manual reading of the strips and genotype detection by comparison with la test does not directly detect hpv52 but combines a set of probes that detects hpv33 , 35 and 58 ( hpv mix ) . specimens that test negative for hpv33 , 35 and 58 individually but are positive for hpv mix are considered to be hpv52 positive . the specimens that test positive for hpv mix and for hpv33 , 35 and/or 58 have an uncertain hpv52 status , for this analysis , these specimens were considered to be hpv52 negative , since coinfection with hpv52 can not be ruled out by this test . the procedure performed into two physically separated areas ( pre - pcr and post - pcr ) in order to avoid contamination of samples with previously amplified products . the reading of the strips , produced by the two methods , was made by one well - experienced biomedical scientist . discrepant interpretations were resolved by a second biomedical scientist and consensus review performed without knowledge of prior results . the la - hpv test does not cross - react with a variety of viruses , bacteria , protozoa and yeast that could be present in cervical specimens . pairwise comparison of amplilute method and magna pure method was performed by using kappa ( ) statistics . a value of 0 indicates no agreement better than chance , and value of 1 indicates perfect agreement , values from 0 to 0.20 , 0.21 to 0.40 , 0.41 to 0.60 , 0.61 to 0.80 and 0.81 to 1.00 indicate poor , fair , moderate , good , and very good strengths of agreement , respectively . receiver operating characteristics ( roc ) curve analysis was applied to calculate and compare amplilute method and magna pure method with cytological findings . in addition roc analysis was applied to calculate and compare amplilute method and magna pure method with histological findings . all the statistical analyses were obtained with the statistical package for the social sciences ( spss ) computer software . a total of 253 women were analyzed in the present study by means of screening for the presence of hpv dna by using two different dna extraction methods . out of the 253 cervical smears , 253 nucleic acid extracts were produced using the amplilute liquid media extraction method and 253 dna extracts were generated using the magna pure automated extraction system . all women referred to colposcopy and if visible lesions were found , they were sampled . the dna extracts were evaluated by the la - hpv genotyping test and compared against the reported cytological and histological diagnoses . the levels of sample adequacy for cytological examination and for nucleic acid extraction and amplification efficiency among the specimens , based on -globin positivity , did not differ dramatically between the tests ( table 1 ) . sample adequacy was higher with amplilute extracts ( 100% ) than with the magna pure method ( 99.6% ) and cytology examination ( 97.6% ) . only one nucleic acid extract generated by the magna pure lc was invalid after la genotyping test due to the absence of high and low -globin result . the comparison of hpv la test results using amplilute extracts with equivalent magna pure extracts showed an overall concordance of 93.3% ( = 0.864 , p < hpv genotype profiles were identical in 228/253 ( 90.1% ) of specimens , including of 103 hpv negative samples and 125 positive samples with identical hpv status detected by both methods . out of 125 cases , the same hpv profile was identified in 73 single infections and in 52 multiple infections . discrepant results observed in 25 samples including 11 cases which were amplilute(+)/magna( ) , 5 cases amplilute()/magna(+ ) , 5 cases identified as single infections in magna but as multiple type infections in amplilute , 3 cases in which multiple infections were detected with different hpv profile between the two methods , and in one case , magna generated an invalid result as opposed to amplilute ( table 3 ) . overall , 94/253 ( 37% ) women were diagnosed with normal cytology , 50/253 ( 19.8% ) were exhibited ascus , 91/253 ( 36% ) were diagnosed with lsil , 5/253 ( 2% ) with lsil but having some positions with hsil , and 7/253 ( 3% ) with hsil . in 6 cases ( 2.4% ) the cytological diagnosis was difficult due to inadequacy of the clinical samples ( tables 4 and 5 ) . hpv positivity detected by amplilute was slightly higher compared with magna pure , 57.3% and 54.9% , respectively . analytically , for the cytological category wnl , hpv positivity observed by amplilute method was 29/94 ( 31% ) , for ascus was 46% , for lsil 77% , for lsil / hsil 100% and for hsil 100% . hpv positivity detected by the magna pure lc method for the aforementioned categories was : 33% , 44% , 78% , 80% , and 100% , respectively , ( tables 4 and 5 ) . within the studied population , in total , single hpv infections detected by the amplilute was 28.5% and by magna pure 30.4% whereas multiple type of infection was observed in 26.5% and 23% , respectively . in this study , the hpv distribution of single infections ( divided into two categories according to the hpv genotype oncogenicity : lr and hr ) as well as multiple infections ( divided into three categories : lr ( when only low - risk hpv types were present ) , hr - lr ( when low- and high - risk hpv types were present ) and hr ( when only high - risk hpv types were present ) in all cytological categories studied are given in figure 1 for the amplilute method and in figure 2 for the magna pure . more multiple infections were detected by amplilute method in all cytological categories compared with the magna pure ( p not statistical significant ) . analytically , for the cytological category wnl , the composition of multiple infections observed by amplilute method was at 6.4% with hr , 4.2% with hr - lr , and 1% with lr , for ascus at 4% with hr , 12% with hr - lr , and 2% with lr , for lsil at 9% with hr , 24% with hr - lr , and 9% with lr , for lsil / hsil at 40% with hr and 20% with hr - lr and for hsil at 40% with hr , 20% with hr - lr . multiple infections detected by the magna pure lc method for the aforementioned categories was wnl at 4.2% with hr , 4.2% with hr - lr , and 2% with lr , for ascus at 4% with hr , 8% with hr - lr and 2% with lr , for lsil at 4% with hr , 23% with hr - lr and 9% with lr , for lsil / hsil at 20% with hr and 20% with hr - lr and for hsil at 14% with hr , 43% with hr - lr . the hpv type prevalence according to the two extraction methods is given below with hpv16 being the most frequent type detected , in both types of infections and by the two methods , followed by hpv31 , hpv53 , hpv6 , hpv33 , hpv45 , hpv42 , and hpv51 as detected by the amplilute method and by hpv31 , hpv53 , hpv18 , hpv51 , hpv18 , hpv6 , and hpv33 as detected by the magna method ( data not showed ) . the two extraction methods were compared against the cytological findings ( inadequate cytological samples were excluded ) . in terms of processing evaluation , amplilute method obtained better results than magna pure method ( amplilute : sensitivity ( se ) = 73.2% , specificity ( sp ) = 69.15% , positive predictive value ( ppv ) = 79.43% , negative predictive value ( npv ) = 61.32% ; magna pure : se = 67.32% , sp = 67.02% , ppv = 76.87% , npv = 55.75% ) . both methods performed well when compared against the cytological diagnosis ; nevertheless , the amplilute method demonstrated a slightest higher area under curve ( auc ) 0.712 ( std . positive histological result was found in 129 cases . from patients with normal cytology , only 13 out of 94 ( 13.8% ) in biopsy had a hpv lesion or cin 1 diagnosis . from patients with ascus , 14 out of 50 ( 28% ) had hpv in biopsy , 8/50 ( 16% ) had cin 1 , whereas only 1 ( 2% ) patient had cin 2 . in lsil , 29/91 ( 31.8% ) had a biopsy diagnosis of hpv , 39/91 ( 42.8% ) had cin 1 , 7/91 ( 7.7% ) had cin 2 , and only 2/91 ( 2.2% ) had cin 3 . all cases classified as hsil with thinprep cytology had a biopsy diagnosis of either cin2 ( 4/7 , 57.2% ) or cin3 ( 3/7 , 42.8% ) . in lsil / hsil category , 1 patient ( 20% ) had cin1 , 3/5 ( 60% ) had cin2 and 1/5 ( 20% ) had cin3 . four patients with no cytological diagnosis , due to inadequate of sampling , biopsy revealed lesions with hpv . results of biopsy reading and hpv genotyping by the two extraction methods are demonstrated in tables 7 and 8 . once more , amplilute method showed greater performance over magna pure ( amplilute : se = 100% , sp = 87.5% , ppv = 98.47% , npv = 100% , fpr = 12.5% , fnr = 0.00% , oa = 98.62% , magna pure : se = 91.47% , sp = 81.25% , ppv = 97.52% , npv = 54.17% , fpr = 18.75% , fnr = 8.53% , oa = 90.34% ) . error 0.018 , 95% ci ( 0.9000.971 ) , p < .001 ) and it was higher compared to auc of magna pure method 0.877 ( std . the la - hpv genotyping test provides a standardized , consistent and rapid means for hpv detection and genotyping . this test provides the capacity to identify 37 individual hpv genotypes within a given specimen and ascertain whether recurrent hpv positivity is , in fact , due to the persistence of a specific hr hpv genotype , meaning a substantially increased risk of disease progression [ 2325 ] . current specimen processing protocols recommend the use of manual extraction of dna using the amplilute liquid media extraction kit , based on the qiaamp method ( qiagen , inc . , this method of dna preparation is time consuming and labor intensive and is prone to potential specimen cross - contamination , particularly when large numbers of specimens are being processed . an alternative method for dna extraction is the automated magna pure lc extraction system , developed by the same company , which could facilitate the assay by minimizing the potential sample - contamination , hands - on time as well as increase labor efficiency and sample accuracy . in the present study , we assessed dna extracts form preservcyt cervical samples , generated by the automated magna pure extraction system and by the manual amplilute method ( both recommended by roche ) for hpv testing using the la - hpv genotyping and detection assays . in addition , we correlated those results with the cytological and histological findings of the enrolled participants . among the 253 thinprep pap tests analyzed in our study , only one extract from the magna pure modified dna - i extraction protocol was found to be invalid due to the absence of low and high -globin . in contrast , all nucleic acids generated from the amplilute protocol were valid for hpv dna genotyping . this marginal difference in sample adequacy could be either due to the high amplilute protocol efficiency or to the variations in aliquoting the specific sample resulting in an inadequacy of cellular material for the automated procedure . comparison of the hpv genotyping results , obtained with the amplilute dna to those from the magna demonstrated a substantial level of agreement ( 93.3% ) , with value of 0.864 . both extraction methods , in terms of qualitative results performed equally well when compared against the cytological diagnosis , with amplilute method demonstrating a small predominance ( auc of amplilute : 0.712 versus auc of magna : 0.672 ) . nevertheless , the amplilute method exhibited higher sensitivity , specificity , positive and negative predictive values as opposed to magna method . the same outcome , amplilute method being more efficient than the magna , was noticed when we compared the two methods with the histological diagnosis . auc of amplilute was 0.935 in contrast with the auc of magna which was 0.877 . hpv types identified in individual samples by each method are largely in agreement 90.1% ( 228/253 ) . in all studied cases , for the former , hpv overall positivity was calculated at 57.3% comprising 30.4% of hpv detected as single infection and 27% as multiple . and for the latter , the respective positivity was 54.5% , 32% as single and 23% as multiple type of infection . the prevalence of hpv - infected samples increased , in both methods , with the severity of cytological diagnoses : 30.8% of amplilute versus 33% of magna in the wnl , 46% versus 44% of ascus , 84.6% versus 77% of lsil , 100% versus 80% of lsil / hsil and 100% by both of hsil . since , there are limited greek epidemiological data available , studies that yielded similar findings in healthy women to our results , were the report by papachristou et al . who found that the corresponding prevalence was 31.5% and agorastos et al . at 36.3% . other studies in our country demonstrated that hpv dna presence in wnl varied from 24% [ 28 , 29 ] to 18% [ 30 , 31 ] with the lowest prevalence reported at 2.5% . this variability is also observed widely in the literature and is mainly due to the different criteria used for selecting the study population and also due to different molecular test applied . the biological meaning out of this is that latent hpv infections with no apparent underlying disease , which would otherwise not be diagnosed on cytological evaluation , are detectable with highly sensitive pcr - based methods . amplilute correctly identified 87.5% of the negative histological cases as hpv negative samples compared to 81.2% of magna . in addition , in cases with histological evaluation from hpv up to cin2 , magna missed 10 cases counting for 8% ( 10/123 ) of the population with these specific histological abnormalities , whereas all those cases were accurately detected as hpv positive samples by amplilute . in 144 cases with cytological findings of wnl and ascus , hpv was detected approximately in 52 cases ( more than 50% of which were hr hpv types ) and from which only 33 participants exhibited histological lesions of hpv up to cin2 . the remaining cases need to be followed up closely due to their elevated risk for developing a high - grade cervical lesion in the future . the small number of cases investigated in this present study limits our ability to conclude correct and representative epidemiological data on hpv prevalence in greek women . nevertheless , data of this report on hpv distribution add to a rich body on literature demonstrating that hpv 16 was the most frequent type detected in both types of infections followed by hr hpv 31 , 53 , 33 , 45 , 18 , and 51 . the observation of hpv 53 being among the three most prevalent hr hpv types detected is consistent with findings of previous greek studies [ 28 , 31 ] . however , the prevalence and clinical role of hpv 51 needs to be clarified through further studies . critical points on multiple infections are succinctly presented , since the detailed analysis of multiple infections identified in the clinical specimens was beyond the scope of this work and they will be discussed analytically on other report . nevertheless , they were highly detected among the hpv positive participants : 47% ( 68/145 ) by the amplilute and 42% ( 58/138 ) by the magna . multiple type hpv infections were identified in approximately 50% of the hpv - infected individuals in wnl category , at 34% in ascus , at 50% in lsil and in lsil / hsil and finally at 60% in hsil category . the elevated incidence rate of multiple infections in our results are in line with the results described by sandri et al . who found multiple infections in 43% of the studied population and by gargiulo et al . in 49.7% . regarding the discordant results observed between the two extraction methods , as showed in table 3 , in 44% ( 11/25 ) of the cases , magna failed in detecting hpv as opposed to the amplilute . ten of these eleven cases were histological confirmed as hpv and correctly identified by the amplilute . in 20% ( 5/25 ) of the cases , which were positive by the magna but negative by the amplilute method , there were either negative in histology or there were with normal cytology and without visible lesion upon colposcopy ( thus for those women , cervical biopsy was not taken ) . in this regard , amplilute method gave a correct negative call and those cases could be considered as magna false positive results . in 32% ( 8/25 ) of cases , which were positive by the two methods but differing in the number of hpv genotypes detected , amplilute demonstrated higher level of detecting additional hpv genotypes in seven cases , apart from the common shared types , as opposed to magna . those extra genotypes detected carry an increased clinical significance , since there were hr genotypes and could alter the clinical outcome of the patient . only in one case , 4% ( 1/25 ) the hpv genotypes were completely different by the two methods and also in one case magna detected one extra genotype than the amplilute . even though the patient population studied does not represent the general population attending our hospital , but only women who agreed to undergo further examination if necessary , the clinical samples tested covered a range of pathologies , from samples that were cytologically normal to samples that had hsil . therefore , the results ( as well as the discordant result rate ) for hpv detection generated by the two extraction methods demonstrated in the present study can be representative of the hpv infection in a screening population . it is important to mention that the decision of utilizing the modified dna protocol for the automated magna pure extraction system , was made based on a recent report . it compares dna extraction efficiencies using the same extraction system with the incorporation of three different working dna extraction kits : ( i ) blood cells high - performance protol ( dna - i kit ) , ( ii ) total nucleic acid ( tna ) kit , and ( iii ) a modified dna - i kit with the manual amplilute protocol for both amplicor and la hpv tests in 150 specimens . although the women enrolled in the above study had histological confirmed cervical abnormalities , no comparison was made between the dna extracts and the hpv genotyping test with their cytohistological findings . we used the modified dna - i kit ( blood cells high - performance protol ) using 1 ml of preservcyt sample as reported by stevens et al . , since it performed better than the other two protocols and it was recommended by the author . at this point , it is important to emphasise that even though for the manual amplilute method we used one fourth of biological material ( 250 l ) as opposed to the automated magna dna - i modified protocol ( 1000 l ) and equal amount of dna extract inputs were used for pcr amplification ( 50 l ) and subsequently hpv detection , more hpv - positive cases were detected by the manual method . someone would assume that increased hpv genotype detection would occur when a bigger amount of clinical sample is incorporated in the dna extraction procedure , since more representative epithelial cells would be present in the sample tested , and thus increasing the possibility of hpv genotypes been detected by the assay used . the findings of our report , which are in contrast with previous reported one , declared the opposite , indicating that the current manual amplilute protocol for dna preparation , provides adequate dna quality , and consequently , it is capable of detecting hpv infections with high sensitivity . having in mind that both methods gave comparative results when tested against cytology and histology , our data provide an additional advantage to amplilute , since reliable results can be obtained even when small volumes of biological material are available for molecular use . in the literature , there is also a report that utilizing the same magna pure automated extraction system , compares the amplicor hpv test to the inno - lipa hpv genotyping test , using only the tna extraction kit for dna isolation for amplicor test , making , thus , difficult the direct comparison with this work . several studies have undertaken assessment of the utility of various automated dna extraction platforms in conjunction with the la hpv test without comparing them with manual extraction methods [ 34 , 36 , 37 ] . moreover , in the literature , there are limited studies that address the variability in hpv genotyping introduced by small changes in front - end dna extraction procedures prior to use in the la hpv genotyping test [ 38 , 39 ] . from those reports , it was interesting found that minor changes to equally valid dna extraction methods appeared to vary the assay 's performance . for example , varying the volume of preservcyt for dna extraction or varying the centrifuge speed during dna extraction or varying the amount of template dna used for amplification can impact assay results . it is well documented and widely accepted that it is difficult to achieve reproducible and accurate hpv genotyping results using pcr - based methods , particularly when individual specimens may contain multiple concurrent infections and/or low viral copy numbers . each of the many steps of testing , from collection of the cervical sample to the final recording of the result , can introduce important variability . large - scale data comparing different methods of dna isolation are needed to reach an optimal protocol for the hpv presence detection and accurate genotyping in order to monitor viral clearance , and most importantly hpv persistence , which is considered as a key factor in cervical cancer development . moreover , accurate and sensitive methods for detection of hpv should be determined , since their performance can strongly affect the results of epidemiological studies and the clinical treatment strategy selected . therefore , the magna extraction method should be tested against other automated and manual nucleic acids isolation techniques and in large population studies before being implemented and routinely used in laboratories . if would be proven accurate in detecting hpv infection , laboratories particularly those involved in large - scale hpv genotyping studies or handling a large amount of clinical specimens or can afford the cost of the automated procedure ( more than two and a half times most pricey than the manual procedure ) could profit from this automated nucleic acid isolation technique . accurate laboratory assays for the diagnosis of hpv infection are being recognized increasingly as essential for clinical management of women with cervical precancerous lesions . the first and most important step in molecular diagnosis of hpv infection is the nucleic acid isolation . an alternative approach to manual extraction procedures , which are time consuming and labor intense , is the automated processing of clinical specimens for hpv detection and genotyping which minimizes the potential sample contamination and the hands - on time . from our data , it was concluded that both dna extraction methods demonstrated similar clinical performance , with no significant difference for any of the outcomes assessed even if for some outcomes the amplilute method exhibited higher sensitivity , specificity positive and negative predictive values as opposed to magna methods . based on the results of this study , the automated nucleic acid isolation method should be tested versus other automated and manual techniques before it is routinely implemented . in addition , additional studies with larger populations are required to be carried out using the automated extraction system in order for its potential value to accurate hpv detection been determined .

in present pilot study aimed to estimate , presence of mycobacterium avium subspecies paratuberculosis ( map ) antibodies in the human serum samples originating from north india using indigenous absorbed elisa kit ( elisa kit ) . the phase i , elisa kit was optimized using protoplasmic antigen from native isolate of map indian bison type recovered from the biopsies of crohn 's disease patients . the phase ii , sensitivity and specificity of the kit were estimated as 40.0 and 83.3% , respectively , when evaluated in 40 human serum samples ( 5 crohn 's disease and 22 ulcerative colitis patients and 13 healthy human subjects ) with defined map status with respect to stool culture . seroprevalence of map antibodies was higher in cd patients ( 80.0% ) as compared to ulcerative colitis patients ( 4.5% ) and normal human subjects ( 15.3% ) . the phase iii , seroprevalence of map antibodies was estimated as 23.4% , on the basis of the screening of 452 human serum samples ( without history ) from different geographical regions of north india . region - wise , 34.0 , 33.3 , 32.8 , 25.0 , 23.0 , 17.7 , and 12.5% samples were positive from the states of punjab , uttarakhand , new delhi , himachal pradesh , haryana , uttar pradesh and jammu and kashmir , respectively . study reported moderately higher presence of map antibodies in human population , which necessitates programs to reduce the bioburden of map in the environment and in animal population .

mycobacterium avium subspecies paratuberculosis ( map ) , the cause of johne 's disease ( jd ) , has emerged as major pathogen of concern for human health worldwide and has also been associated with crohn 's disease ( cd ) in human beings [ 13 ] . cd is a chronic incurable inflammatory bowel disease ( ibd ) of gastrointestinal tract ( git ) involving mesenteric and regional lymph nodes and resulting in chronic segmental inflammation that most commonly involves distal ileum or proximal colon , though lesions can occur at any location throughout the git . association of map with cases of cd has been supported by frequent isolation of map in significantly higher number of cd patients than patients with other bowel disease syndromes and healthy controls [ 2 , 3 ] . studies have also shown that like animal paratuberculosis , map infection in humans is systematic [ 3 , 4 ] . pcr , in situ hybridization , and other molecular tools successfully detected map dna in the tissues and blood samples of cd patients [ 57 ] . immunological studies using specific , highly purified recombinant antigens also supported the association between map infection and cases of cd [ 810 ] . in the developed countries , commercial elisa kits employed for the detection of map antibodies in animals have been successfully adopted for the screening of human serum samples [ 11 , 12 ] . indigenous elisa kit , developed in india , was significantly superior when compared with imported commercial elisa kits for the screening of animals [ 13 , 14 ] . recently , singh et al . reported high prevalence of map in the animal health care workers and cd patients . however , in view of the lack of indigenous diagnostic kits and reagents , information on the prevalence of map in ibd patients ( consisting of ulcerative colitis and crohn 's disease ) and 1.2 billion human population of the country is limited . the study employed indigenous absorbed elisa kit , based on protoplasmic antigen from native map genotype recovered from biopsies of cd patient ( a 46 ) , for the estimation of sero - prevalence of map antibodies in the human population of north india . the study was conducted in three phases . semipurified protoplasmic antigen ( pa ) strain ( a46 ) of map recovered from the biopsies of cd patient in fourth passage level . map was subpassaged in 710 slants of hey medium with mycobactin j at 37c for 8 months . growth was harvested , washed and sonicated at 100 w ( 15 hz ) for 20 min in ice slurry giving 20 cycles of 30 s rest . sonicate was centrifuged at 9727 g for 30 min at 4c using biocentrifuge . supernatant was dispensed in aliquots of 0.5 and 1 ml and stored at 20c till further use . a portion of aliquots was used for protein measurement as per lowry et al . . presently , country lacks indigenous kits , for the screening of either animal or human serum samples . in the present study , standardized as per milner et al . was employed . optimum concentration of antigen , serum , and second antibody ( conjugate ) was determined by checkerboard analysis ( pa 0.1 g per well ; serum dilution 1 : 50 ; rabbit anti - human horseradish peroxidase 1 : 8000 ) . to reduce the background colour and nonspecific binding of protein , optimum blocking agent concentration was standardized using signal - to - noise ratio . since skimmed milk ( 3% ) in pbs had highest noise ratio , therefore it was employed as blocking agent in the indigenous absorbed elisa kit . the 0.1 g of semipurified protoplasmic antigen ( pa ) in 100 l of carbonate bicarbonate buffer ( ph 9.6 ) was used for coating duplicate wells of flat - bottom 96 well elisa plate ( grenier bio - one ) . plates were incubated overnight at 4c followed by washing thrice with washing buffer , pbst ( pbs with 0.05% tween 20 ) . blocking was done by 200 l of 3% skimmed milk in pbs and incubated at 37c for 1 h. after incubation , plates were washed thrice with pbst and then 100 l of test serum , preabsorbed in pbst containing 1% bsa and 2 mg / ml mycobacterium phlei for overnight at 4c as per method of klausen et al . , were added to duplicate well and incubated for 2 h at 37c . after incubation , three washings ( 5 minutes each ) were given with pbst , and 100 l of optimally pbs - diluted ( 1 : 8000 ) conjugate ( sigma ) in was added to all the wells . plate was incubated for 1 h at 37c and then washed three times with pbst . finally , 200 l of freshly prepared substrate ( ortho - phenylene diamine dihydrochloride-(opd ) ) , 5 mg per plate in substrate buffer ( ph 5.0 ) , was added to each well . following incubation ( in dark ) for 20 min at room temperature , absorbance was read at 450 nm in elisa reader without the addition of stop solution ( 5 n h2so4 ) . blank , positive , and conjugate controls were also run along with serum samples in each plate . od values of test samples were transformed to s / p ratio as per collins , and samples in positive ratios , corresponding with strong positive category of collins , were considered as positive for map infection . sensitivity and specificity of the kit were evaluated as per arizmendi and grimes using 40 human serum samples ( 5 cd and 22 uc patients and 13 healthy human subjects ) with known map culture record . samples from cd and uc patients were collected from the department of gastroenterology , all india institute of medical sciences ( aiims ) , new delhi and asopa hospital and research center , agra , up , india while samples from healthy human subjects were from blood donors at aastha pathology laboratory , agra , up , india . performance of the kit with respect to stool culture was compared by calculating kappa scores ( proportional agreement ) as per altman ( 0 < , poor ; 0.00.20 , slight ; 0.210.40 , fair ; 0.410.60 , moderate ; 0.610.80 , substantial and 0.81100 , almost perfect ) . serum samples collected randomly from 452 human beings ( 329 males and 123 females ) in the northern region of the country , between 2004 and 2008 , were screened for the map antibodies . geographically , serum samples were obtained from human beings belonging to the states of uttar pradesh ( 180 ) , rajasthan ( 47 ) , jammu and kashmir ( 16 ) , uttra - khand ( 12 ) , himanchal pradesh ( 2 ) , punjab ( 50 ) , haryana ( 65 ) , and new delhi ( 70 ) in north india . these serum samples were obtained from human subject without known disease history for map antibodies and were therefore considered normal . serum samples were screened using indigenous absorbed elisa kit . for the reproducibility of the results and to reduce well - to - well and plate - to - plate variations , all tests were performed using four known positive and three negative control serum samples obtained from the earlier studies . to estimate sensitivity and specificity of indigenous absorbed elisa kit , culture and elisa tests were compared on stool and serum samples of 40 human beings . the 15.0 ( 6 ) and 12.5% ( 5 ) samples were positive exclusively in culture and elisa kit , respectively . there was substantial perfect agreement ( proportion agreement = 0.72 , kappa value = 0.24 ) between the two tests . of the 40 human serum samples ( cd and uc patients and normal human beings ) used in optimization of indigenous absorbed elisa kit prevalence of map was higher ( 18.5% ) in inflammatory bowel disease ( cd and uc ) patients as compared to normal human subjects ( 15.3 ) . however , individually , 80.0 , 4.5 , and 15.3% serum samples were positive from cd and uc patients and normal human beings , respectively ( table 1 ) . of the 452 human serum samples screened from north india , 23.4% were positive for anti - map antibodies ( table 2 ) . seroprevalence of map antibodies was higher in females ( 31.7% ) as compared to male population ( 20.3% ) in north india . state - wise , the highest seroprevalence was found in samples from punjab ( 34.0% ) , followed by uttarakhand ( 33.3% ) , new delhi ( 32.8% ) , himachal pradesh ( 25.0% ) , haryana ( 23.% ) , uttar pradesh ( 17.7% ) , and jammu and kashmir ( 12.5% ) . in the present study , indigenous absorbed elisa kit based on native isolate of map of human origin was standardized to detect map antibodies in ibd ( cd an uc ) patients and to estimate seroprevalence of map infection in human beings from north india . elisa was a rapid and cost - effective alternative to organism detection and can detect low concentration of antibodies in early and late stages of the map infection . optimum antigen concentration was 0.1 g / well as compared to 1.0 g / well used by molina et al . . in the present study , 1 : 50 serum dilution was used as compared to 1 : 25 ( lower ) serum dilution used by molina et al . for the detection of map antibodies in animals . the use of lower concentration of native antigen and higher dilution of serum in the indigenous absorbed elisa kit may be due to the higher pathogenicity of indian map strain ( indian bison type ) in animals . to reduce background noise of proteins , 3% skimmed milk was found to be optimum blocking agent as compared to 5 and 10% skimmed milk and 3 , 5 , and 10% bovine serum albumin . johnson et al . also reported the superiority of skimmed milk as blocking agent in comparison to bsa . cutoff value was calculated by sample - to - positive ( s / p ) ratio , instead of average od of negative samples 2 sd [ 15 , 18 , 22 ] . several researchers advocated quantitative evaluation of od values or s / p ratios , rather than relying solely on positive or negative classifications defined by a single cutoff value [ 30 , 31 ] . studies reported relatively consistent increase in likelihood ratios as the optical density and s / p ratios increased relative to both concurrent fecal culture result and known infection status [ 32 , 33 ] . other researchers have also used s / p ratio as cutoff value for the detection of map antibodies in human [ 11 , 12 ] and animals [ 16 , 31 ] samples . the presence of map antibodies was higher in cd patients ( 80.0% ) as compared to uc patients ( 4.5% ) and normal human subjects ( 15.3% ) using indigenous absorbed elisa kit , other researchers [ 10 , 11 ] have also reported higher presence of map in cd patients as compared to uc patients and control subjects . the lower presence of antibodies in uc patients as compared to healthy controls may be attributed to the altered immune response due to the use of immunosuppressive drugs for their treatment . unlike present findings , 35.0% seropositivity rates for all the groups in a population - based study were reported by bernstein et al . and there was no difference in the rate of positive samples of cd and uc patients , and healthy controls or nonaffected siblings . sensitivity and specificity of indigenous absorbed elisa kit with respect to stool culture were 40.0 and 83.3% , respectively . slightly lower specificity of the elisa kit may be attributed to the presence of cell - wall - deficient ( cwd ) forms of map bacilli in human beings . indigenous absorbed elisa kit had comparable sensitivity and specificity with respect to imported commercial elisa kits when employed to screen serum samples of cattle , goat , and sheep origin ( unpublished data ) . therefore , indigenous absorbed elisa kit may be employed for the screening of anti - map antibodies in humans and animals . the new indigenous elisa kit facilitated the screening of large number of human serum samples , and seropresence of map antibodies in human beings from north india was 23.4% . a previous study reported 38.0% prevalence of map in human subjects from agra region using indigenous un - absorbed elisa kit . marginally lowered prevalence of map antibodies in the present study may be due to the pre - adsorption of test serum with m. phlei . female subjects ( 31.7% ) had higher presence of map antibodies as compared to male subjects ( 20.3% ) . this may be attributed to the poor sanitation , lowered nutritional status , and additional physical stress ( pregnancy , parturition , lactation , etc . ) on females , as reported in animals . therefore , high prevalence of map in human beings may be due to the higher bioburden of map in the environment and/or the presence of map in the food chain [ 19 , 37 , 38 ] . bernstein et al . also reported higher ( 33.6% ) seroprevalence of map in the population of manitoba by adapting . indicated that manitobans are simply more exposed to map than population of north india or that they have a different genetic ability to mount a response to the infection . within states in india , seropresence of map in human beings ranged from 12.5 to 34.0% ( table 1 ) . higher sero - presence of map antibodies may not be conclusive for map infection in the human population of north india . however , the detection of map antibodies in higher numbers of human samples indicated higher exposure of human population to map infection . therefore , the control of map infection in source population ( animals ) , will be required to reduce the exposure of human population .

we present a method based on self - assembling oligonucleotides to anchor proteins to a supported membrane surface . this anchoring method allows control of the surface density of multiple proteins . by incorporating additional recognition sequences into the dna linkers , defined heterodimers can be produced upon the addition of a heterospecific dna cross - linking strand . characterization by fluorescence cross - correlation spectroscopy ( fccs ) confirmed lateral mobility and the formation of specific heterodimers . we further demonstrate that proteins linked in this manner as either monomers or dimers can form functional interfaces with living cells .

numerous biological processes , including immune recognition , animal development , and the misregulation of development in cancer cell progression , involve signaling interactions across cell cell junctions . in this juxtacrine configuration , ligands and receptors bind to each other from apposed cell surfaces . supported lipid membranes can reconstitute functional juxtacrine signaling interfaces with living cells and have been a useful tool to study and manipulate these interactions . protein ligands that would naturally occur on one cell surface are instead synthetically coupled to the supported membrane . the lateral mobility of the supported membrane enables these ligands to diffuse and assemble into functional clusters as they engage their cognate ligands on the adjacent live cell surface . such signaling clusters are emerging as a general phenomenon common to many juxtacrine signaling interactions . recent studies on the eph and egfr families of receptor tyrosine kinases ( rtks ) indicate that heterooligomerization of proteins within signaling clusters may exert additional layers of regulatory control . the increasing numbers of therapeutic bispecific antibodies entering clinical trials suggest that it may be possible to modulate signaling cluster content with therapeutic benefit . with the goal of extending the utility of supported membranes for the study of complex , multicomponent clusters and the demonstrated success of dna - based protein assembly , we report here a dna - based assembly strategy to associate proteins with membranes and to control their assembly into defined heterodimers or higher - order oligomers . thiol - functionalized dna was coupled to maleimide - functionalized supported membranes . unlike strategies that incorporate long alkyl chains onto dna during solid - phase synthesis , this strategy permitted dna conjugation to preformed membranes . in this study , supported membranes with a 1:20 molar ratio of maleimide functionalized phospholipids to 1,2-dioleoyl - sn - glycero-3-phosphocholine were used . the supported membrane was prepared in phosphate buffered saline ( pbs , 10 mm phosphate buffer , 150 mm nacl ) by vesicle deposition on clean glass , as described in the supporting information ( si ) . attachment of the dna and lateral fluidity of the membrane were confirmed by fluorescence recovery after photobleaching ( frap ) after treating the supported membrane with 20 nt single - stranded dna ( ssdna ) bearing a 5-thiol modifier and a 3-6-carboxyfluorescein ( fam ) moiety ( figure 1a ) . ( a ) a representative frap characterization of a supported membrane with fluorescently labeled ssdna is shown . ( b ) the surface density of coupled dna can be varied over a large concentration range . excitation light is interleaved to allow time - resolved data collection , as shown in the graph , and thus removal of the contribution of fluorescence signal bleedthrough to cross - correlation . ( b ) fccs analysis indicated the codiffusion of the dna - bound fluorophores , suggesting that they had formed a heterodimer . ( c ) the table compares the relative cross - correlation amplitudes to the lower of the two autocorrelation amplitudes , including positive and negative control samples ( shown in si , figure s5 ) . the surface density of conjugated dna was quantified using membrane standards with known lipid fluorophore surface densities ( figure s1 in the si ) . conjugation of 20 nt and 41 nt ssdna to supported membranes and hybridization of tex615 or alexa fluor 488 ( af488 ) labeled complementary strands to the resulting samples allowed quantification of the dna on the membrane surface by comparing the observed fluorescence intensities of the dna samples to those of the membrane standards . surface density of dna increased proportionally to the concentration of thiol dna that was applied ( figure 1b ) . with incubation concentrations of dna in the low micromolar range , surface densities in the range 03000 strands/m were observed . addition of thiol dna in concentrations above 6 m resulted in further increases in measured surface density ; however , larger variations between identical samples were observed , and the relationship between surface density and incubation concentration became nonlinear . ssdna , 41 nt , coupled to the supported membrane less efficiently than 20 nt ssdna under the same conditions ( pbs , ph 7.4 ) , but the use of ph 8.5 borate buffered saline ( bbs , 10 mm borate , 150 mm nacl ) as a higher ph buffer yielded surface densities of 41 nt ssdna very similar to those of 20 nt ssdna coupled at the lower ph ( figure 1b ) . for the assembly of more complex structures , we designed dna heterodimers using previously published assembly sequences . several strand configurations were evaluated , as shown in si , figure s3 . of these , a particularly successful strategy for the formation of four - strand the two arms of the branched structures were labeled with green and red fluorophores , as shown in figure 2b , allowing characterization by two - color fluorescence cross - correlation spectroscopy ( fccs ) with pulsed interleaved excitation ( pie , see figure 2a and experimental diagram in si , figure s2 ) . fccs has been used to characterize binding characteristics of biomolecules , enzymatic activity , and clustering in cell membranes . pie eliminates artifactual cross - correlation from fluorescence spectral bleed - through by exciting the sample with interleaved laser pulses . the red peak is broad since pulsing is achieved through electro - optic modulation of a continuous wave krar laser . the amplitude of the cross - correlation function is proportional to the concentration of dual - labeled species . measurement of this parameter can be obscured by a variety of artifacts that can both raise or lower the measured cross - correlation amplitude . using control samples that establish the upper and lower bounds of the cross - correlation measurement enables calibration of the cross - correlation signal and quantification of the amount of heterodimer formed ( si and figure s5 ) . performing this analysis of the data shown in figure 2c provides an estimate of 5260% yield of assembled heterodimer . formation of protein heterodimers was demonstrated by assembling heterodimers of fab fragment dna conjugates , effectively reconstructing membrane - bound antibodies . fab fragments can be generated from igg antibodies , which are readily obtained against many proteins . for this study , f(ab)2 fragments generated from polyclonal donkey anti - mouse antibodies were obtained from a commercial source , labeled with fluorophores , and partially reduced with 2-mercaptoethylamine ( 2-mea ) to produce fab fragments with free thiol groups at the c - terminal regions , figure 3a . the products were thoroughly desalted and treated with maleimide - functionalized 20 nt ssdna ( see si for procedures and figure s6 for maldi - tof ms characterization data ) . the conjugates were purified by size exclusion chromatography ( figure 3b ) and analyzed by gel electrophoresis ( sds - page , figure 3c ) . separation of the proteins from free ssdna is shown in the chromatogram ( figure 3b ) . after treating the fab fragments with maleimide dna , gel electrophoresis analysis indicated a species with higher molecular weight compared to the unmodified fab fragments ( figure 3c ) . these conjugates were prepared with different sequences of dna and labeled with distinct fluorophores so that a heterodimer could be prepared on dna - functionalized supported membranes , as shown in figure 3d . the resulting structure was then analyzed by fccs to measure heterodimerization ( figure 3e ) . by comparing the cross correlation amplitude to that of a doubly labeled control sample , we determined that a 4244% assembly yield was obtained for the heterodimeric structure . when expanded to antibody fragments with different specificities this technique provides a way to colocalize two different receptors using a convenient synthetic protocol . ( 2 ) fab before removal of the 2-mea reducing agent . ( 3 ) fab after removal of the 2-mea reducing agent . ( 4 ) fab treated with maleimide dna . ( 5 ) highest molecular weight peak from sec chromatography ( blue shading ) , and ( 6 ) the intermediate molecular weight peak from sec ( orange shading ) . the entry r in the table indicates that the reagent has been removed , ( d ) fccs analysis confirmed formation of a fab heterodimer using the pooled fractions . evaluation of nonspecific interactions between dna functionalized membranes and living cells and accessibility of the dna to presented cells was performed by modification of live jurkat t - cells with surface ssdnas , as described previously . cells were incubated with membranes functionalized with ssdna sequences that were either complementary or noncomplementary to the cell surface ssdna and a membrane of identical composition , but with no dna functionalization . upon washing , cells bound only to the membranes functionalized with complementary ssdna . in addition , only one layer of cells was visible on the sample containing complementary dna , while out - of - focus cells were visible in all samples before rinsing . ( a ) nonadherent jurkat t - cells functionalized with ssdna attached only to membranes functionalized with complementary dna strands . few bound cells were observed on a maleimide - capped sample that lacked dna . ( b ) the dna anchored ephrina1-yfp - his10 construct stimulated mda - mb-231 cells . in these images , epha2 was stained with an antibody after cell permeabilization and imaged with tirf microscopy . ( c ) heterodimeric protein complexes of egf and an inert fab fragment remain intact during interaction with mda - mb-231 cells . presentation of a functional ligand for a cell surface receptor confirmed that membrane - anchored dna is a useful anchor for protein presentation to live cells . ephina1-yfp - his10 , which stimulates the epha2 receptor when presented from supported membrane , was linked to nta3dna . fluorescence signal from the yfp portion of the protein dna conjugate confirmed the presence of the protein and frap analysis confirmed lateral mobility of the anchored protein . mda - mb-231 cells were incubated with the ephrina1-functionalized bilayers for 1 h , fixed with formaldehyde solution , and stained with an anti - epha2 antibody . analysis by total internal reflection fluorescence ( tirf ) microscopy , which illuminates only the interface between the cell and the substrate , showed colocalization of the membrane - bound epha2 receptors with ephrina1 , as expected from previous reports using biotin the ability to direct molecules into signaling clusters was demonstrated using epidermal growth factor ( egf ) presented to mda - mb-231 cells . while egf is typically a soluble ligand , presentation to cells from a membrane surface results in visible clustering of the ligand . upon conjugation to cy3-labeled dna ( figure s8 in si ) , hybridization of the conjugate to dna functionalized membranes , and presentation to mda - mb-231 cells , clustering of egf and phosphorylation of egfr were observed ( figure 4c ) . a fab fragment that has no binding target on the cell membrane presentation of a heterodimer of these molecules resulted in clustering of both , and no evidence of disruption of receptor phosphorylation was observed . these observations demonstrate that colocalization between anchored molecules can be directed independently of any inherent propensity of these molecules to colocalize . we plan to use directed dimerization to study the effect of signaling cluster composition on epha2 signaling . epha2 has been observed to interact with other receptors in the eph and egfr families of rtks . to provide extra stability to the ephrina1dna conjugate , we designed , expressed , and purified an ephrina1snaptag fusion and conjugated this molecule to dna ( figure s9 in si ) , a strategy also used in other studies . egf and ephrina1 dna conjugates were presented to mda - mb-231 cells , which express both egfr and epha2 ( figure 5a ) . while the size of a dna heterodimer is well below the diffraction limit , differences in colocalization between samples containing monomeric ligands and samples containing dimeric ligands could be observed ( figure 5b ) . analysis of fixed cells shows a clear difference in colocalization ( figure 5b , see si and figure s10 for a description of data analysis ) between monomer and heterodimer presented samples . since variations in the ligand distribution can be seen , we also expect to see differences in receptor distribution between cells presented with monomeric ligand and those presented with dimeric ligand . when staining the cells with an antibody against phosphorylated tyrosine 1173 ( py - egfr ) on the egfr receptor , both colocalization measurements decrease , which is likely caused by incomplete antigen staining and some amount of nonspecific binding . colocalization between the ephrina1 ligand and the egfr receptor is considerably increased in cells presented with a heterodimer , demonstrating that the egfr is binding ligand and recruiting the ephrina1 molecule . this observation suggests that the heterodimer is able to interact with the egfr receptor and that ligand receptor binding is preserved . a slight decrease in egf egfr colocalization is observed in the heterodimeric sample , relative to the monomeric control , suggesting that the interaction is somewhat inhibited . additionally , we are are working to purify intact complexes from cell lysate by immunoprecipitation . alteration of receptor localization in cells . ( a ) mda - mb-231 cells were deposited on supported membranes containing dna bearing monomeric or dimeric ligand . ( b ) tirf microscopy analysis demonstrates that the ligands appear segregated when presented as monomers but colocalized when presented as dimers . colocalization was measured as the correlation coefficient in the ephrinal clusters and is described in more detail in the si . ( c ) immunofluorescence staining of ptyr-1173 residue on egfr of the cells in ( b ) shows that the receptor localization is altered by presentation of these ligands . the scale bar represents 10 m , and the error bars depict the standard error of the mean . numbers inset in the example images denote the actual correlation coefficient of the two channels shown in that particular image . we have demonstrated the ability of dna anchors extending from supported membranes to present functional protein ligands to cells and have also shown that this strategy is capable of generating heterodimeric structures that can direct the molecular composition of cell membrane receptor clusters .

parkinson 's disease ( pd ) and tourette 's syndrome ( ts ) lead to important motor disorders among patients such as possible facial amimia in pd and tics in tourette 's syndrome . under the grounded cognition framework that shows the importance of motor embodiment in emotional feeling ( niedenthal , 2007 ) , both types of pathology with motor symptoms should be sufficient to induce potential impairments for these patients when recognizing emotional facial expressions ( efe ) . in this opinion paper , we describe a theoretical framework that assumes potential emotional disorders in parkinson 's disease and tourette 's syndrome based on motor disorders characterizing these two pathologies . we also review different methodological barriers in previous experimental designs that could enable the identification of emotional facial expressions despite emotional disorders in pd and ts .

a prominent view of cognitive and emotional processes assumes that conceptual knowledge emerges from bottom - up perceptual processes to conceptual and a modal symbolic systems . an alternative view has been proposed under the theoretical framework of theories of grounded cognition [ 24 ] suggesting that associative , limbic , and sensorimotor functional components are automatically reactivated for access to specific conceptual knowledge . this theoretical view assumes that disturbing motor processing can induce recognition impairment at the perceptual level . for instance , strack et al . have shown that participants holding a pen between their lips to inhibit smiling , or holding the pen between their teeth to facilitate smiling ( figure 1 ) , produced significant emotional modulation with respect to the funniness of cartoons ( depending on the group of inhibited facial muscles ) . in what follows , we argue that consistent with the grounded cognition theory , motor disorders characterizing different psychopathologies ( parkinson 's disease and tourette 's syndrome ) could be sufficient to induce disturbances in emotion processing . different neural structures have been identified as playing an important role in grounded cognition . for example , these type of links between motor systems and action understanding had previously been developed in the literature on mirror neurons , first shown in monkeys [ 6 , 7 ] and more recently among humans where premotor and parietal areas were identified as important neural structures for mirror neuron system [ 68 ] . cortical mirror neurons were interpreted as automatic neural processes involved in basic and implicit comprehension of action by other individuals [ 6 , 7 ] . in a similar line of research , wicker et al . have shown that mirror neurons at the level of the insula may support both the feeling and the perception of disgust . this finding suggests that mirror neuron areas dedicated to the functional integration of not only motor but also emotional processes could be widely spread across different high - level cortical areas . among these high - level cortical structures , for example , the right hemisphere cortex seems to be particularly involved in the processing of recognition of emotional expressions , and lesion studies specifically implicate somatosensory cortex . in an attempt to shed further light on the function of these anatomical structures , adolphs et al . carried out a quantitative study involving a detailed analysis of 108 subjects with focal brain lesions . results show a major implication of right somatosensory cortices , including the right anterior supramarginal gyrus , the lower sector of s - i and s - ii , the insula , and the left frontal operculum for recognizing the six basic emotional facial expressions . to a lesser extent , and consistent with previous studies , lesions of the right visual - related cortices also induced a deficit in recognizing specific facial expressions and especially fearful expressions . moreover , adolphs et al . found complementary results showing that lesions that include the right inferior parietal cortex induced impairments in the recognition of negative emotions ( especially fear and sadness ) . consequently , they proposed that right somatosensory cortex generate a representation of the feeling state , which is sometimes used for recognition . it is important to note that the structures involved in recognizing basic emotional facial expressions seem slightly different from those involved in the conceptual knowledge of the emotions showing the involvement of right somatosensory - related cortices ( including s - i and s - ii ) , the insula , and supramarginal gyrus . access to conceptual knowledge was investigated by the authors by means of a task requiring the sorting of efe photographs into piles according to the similarity of the emotion displayed . it can be argued that such a task does not necessarily require conceptual knowledge ; rather , the assessment of perceptual similarity could be sufficient to perform the given task . these differences between the goals and functions of somatosensory cortices versus parietal or temporal cortical areas illustrate the difference between the feeling of and the perceptual recognition of a specific emotional expression . further studies in psychology and cognitive neuroscience have to carefully differentiate between these psychological tasks . subcortical structures might also play a central role in the integration of sensorimotor , limbic , and associative inputs . yelnik and colleagues have shown that different neural structures constituting the basal ganglia ( for instance , the caudate nucleus , the striatum , and of course , the subthalamic nucleus ) integrate the sensory - motor , associative , and limbic functional components ( figure 2 ) . this suggests that motor disorders , induced by subcortical dysfunctions at the level of the basal ganglia , could have either a direct or an indirect effect on emotion processes such as the recognition of efes through the impairment of motor processing . parkinson 's disease ( pd ) is characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta leading to motor as well as associative or addictive disorders [ 1416 ] . the motor disorders characterizing pd are produced by the modulation of neural activity in striatal structures innervated by neurons in substantia nigra pars compacta . the dopaminergic depletion produced by the degeneration of substantia nigra pars compacta induces functional hyperactivity of the subthalamic nuclei and consequently leads to motor disorders that could be modulated by l - dopa therapy . moreover , bilateral deep brain stimulation of the subthalamic nuclei ( stn - dbs ) is often used as a therapy for these patients . stn - dbs is a well - documented and efficient treatment for severely disabled parkinson 's disease ( hereafter , pd ) patients with intractable motor complications . although there is compelling evidence for the clinical efficiency of stn - dbs on motor symptoms [ 18 , 19 ] , there is still an ongoing debate on the effects of stn - dbs on behavioral and emotional manifestations . on one hand , biseul et al . showed that stn - dbs may produce significant emotional impairment specifically in the recognition of fear . however , on the other hand , dujardin et al . reported a more extensive impact of stn - dbs for pd patients concerning the recognition of emotional expressions . these results raise the hypothesis that both l - dopa and stn - dbs therapies impact on motor as well as emotional behavior . however , in addition to these emotional troubles induced by central disorders , other emotional disorders might indirectly be induced by peripheral dysfunction . among the different motor disorders of which pd patients suffer , amimia often appears as a consequence of motor symptoms in pd . this motor problem may have consequences for subsequent emotional processes . as proposed elsewhere under the framework of grounded cognition [ 2 , 3 , 22 , 23 ] the facial amimia suffered by pd patients induces more severe and long - term deficiencies than temporarily holding a pen in one 's mouth during the course of an experiment . we believe that motor disorders may induce a wide range of emotional impairments that could be demonstrated even through emotion recognition tasks . as is pd , tourette 's syndrome ( ts ) is characterized in part by motor disorders . whereas pd patients often suffer from facial amimia , ts patients often have facial tics . more precisely , ts is a neuropsychiatric syndrome defined by chronic multiple motor and vocal tics sometimes accompanied by anxiety or obsessive - compulsive disorders ( ocd ) . as suggested by mink , basal ganglia and frontocortical dysfunctions probably constitute the root of ts physiopathology ( see also for a review ) . proposed that neurophysiological and neuropsychological data on ocd highlight abnormalities in frontostriatal regions that may potentially mediate correct recognition of emotional expressions . albin and mink suggest a dopaminergic dysregulation that could be at the root of the tourette 's syndrome . this hypothesis is based on biochemical analyses of postmortem striatum from ts patients revealing a significant elevation in the number of dopamine uptake carrier sites [ 29 , 30 ] . the hypothesis is also derived from animal data that shows that the injection of a dopaminergic agonist increases the production of motor stereotypy very similar to ts in treated animals . moreover collectively , such dopaminergic sensitivity in ts could be sufficient to induce motor dysfunction ( verbal but also motor tics ) . theories of grounded cognition extended to account for emotion processing suggest that motor disorders might produce deficits in the processing of emotional information , as has been previously suggested for parkinson 's disease . that is , as regards parkinson 's disease , dopaminergic dysfunction in the basal ganglia might induce compromised processing of motor functions with consecutive consequences for emotional information processing . based on the embodiment theory , the motor disorders characterizing tourette 's syndrome ( motor hyperactivity and tics ) and parkinson 's disease ( dyskinesia ) should be sufficient to produce emotional disturbance . as suggested by previous findings ( see also for a review ) , motor dysfunction at a peripheral level is sufficient to induce emotional modulation at the central level . moreover , recent debate shows that such consequences of the embodiment theory on emotional processes could be very fast and beyond the scope of consciousness [ 3335 ] . however , such potential influence of dopaminergic dysfunction on emotional processes through motor disorders could be accompanied by other problems directly affecting the central level . concerning parkinson 's disease , it has been shown that dopaminergic depletion has important consequences at the motor level because of the hyperactivity of the subthalamic nuclei . however , as reported by yelnik et al . in other words , dopaminergic depletion could also have a major influence on the limbic and associative functional components indirectly through motor disorders at a peripheral level but also directly at the level central nervous system through subthalamic nuclei hyperactivity . moreover , dopaminergic depletion may have functional consequences on other neural structures at the level of the basal ganglia , and more specifically on the limbic components of these subcortical structures ( as well as related cortical areas ) . this implies that dopaminergic depletion could have not only a direct influence on emotional processes occurring at the central level but also an indirect influence , based on the embodiment theory , for example , through facial amimia observed at the peripheral level . similar conclusions can be drawn for tourette 's syndrome . a dopaminergic hypersensitivity as proposed by or might have important consequences on emotional processes occurring at the level of the basal ganglia . the pallidum , a subcortical neural structure potentially involved in ts , could have direct implications on emotional processes in a manner similar to subthalamic nuclei in pd . as for pd patients , dopaminergic modulation probably has a direct influence on basal ganglia ( and , therefore , subcortical limbic processes ) combined with an indirect influence through the well - established motor disorders observed in ts ( still under the theoretical framework of the embodiment theory ) . however , despite these strong theoretical assumptions of emotional disorders in pd and ts , literature has brought into light controversial evidence concerning emotional impairments that recommends to carefully take into account methodological considerations . an examination of the literature on emotional processing in pd yields a set of controversial findings . for instance , sprengelmeyer et al . reported that recent untreated pd patients presented significant efe impairments ( more specifically on disgust ) as compared to healthy controls but this difference was not observed on more severe but treated pd patients . using an innovative technique allowing to take into account the difficulty of recognizing one or another efe , the authors reported significant impairments on disgust efe . however , using an experimental procedure for recording accuracy in categorizing efe together with a rating scale of the intensity of each efe , dujardin et al . they found that pd patients were less accurate than healthy controls in decoding angry , sad , and disgusted efes . moreover , pd patients rated efes other than anger , sadness , and disgust as less intense than healthy controls . more surprisingly , pd patients rated the surprise efe more intensely than the healthy control group . generally , these last findings suggest that parkinson 's disease patients may have more widespread emotional impairments than previously reported in literature , but the experimental tasks habitually undertaken were not sensitive enough to reveal these impairments . in other words reported interesting data showing that a patient with severe amygdala lesion inducing a strong deficit in the feeling of fear was nonetheless able to recognize in a reliable manner fearful expressions by looking at specific perceptual details in the face such as the opening of the eyes . this experiment illustrates the fact that participants may use perceptual details , even without a clear emotional feeling of the different efes , to recognize efes . in order to decrease the influence of perceptual strategies on the performance of efe tasks , we advocate the use of rapid presentations of emotional stimuli . rapid presentation of critical stimuli , such as efes , reduces the use of perceptual strategies that can mask the existence of specific emotional deficits such that they go undetected in studies with longer presentation durations [ 21 , 36 , 37 ] . in their examination of tourette 's syndrome , sprengelmeyer et al . found that ts patients with ocd have a specific impairment in recognizing disgusted faces . they showed different distributions of brain activation after disgust - inducing visual stimulation for ocd patients compared to control participants whereas activation after threat - inducing stimulation in ocd participants induced a similar pattern to that observed in control participants . . failed to replicate the finding of sprengelmeyer et al . and found only a disgust impairment for one ts patient with severe ocd . however , as for pd patients , the sensitivity of the task may not be sufficient to show clear evidence of emotional impairment . in both sprengelmeyer et al . and parker et al . , ts participants were exposed to morphed stimuli ( from one efe to another one ) across the emotion hexagon of the 6 basic efe produced by ekman and friesen 's . however , a potential disadvantage is that such repeated exposure to the morphed stimuli across the hexagon significantly improves the perceptual participant 's ability to categorize the efes , and this might be sufficient to induce a ceiling effect . more importantly , long and repeated exposure durations ( 1000 to 5000 ms stimulus duration ) could enable the participants to use cognitive strategies based on specific perceptual features to perform the task . as for pd experiments , these studies illustrate the fact that participants may use perceptual details , even without a clear emotional feeling of the different efes , to perform the task . , we can assume that the motor disorders of which pd and ts patients suffer probably imply important consequences at the emotional level , through the disruption of the embodied processing of emotional events . so far , based on the experimental designs used in previous simulations , we can assume that the absence of reliable results showing such deficits could probably be due to perceptual strategies potentially compensating for the emotional troubles of the patients . therefore , further research will have to ( i ) show such emotional deficits based on more implicit measures of emotional feelings and ( ii ) differentiate between deficits induced by dopaminergic dysregulation occurring at the central level and embodied processing disorders occurring at the peripheral level .

a five - month - old boy who had undergone previously transcatheter balloon atrioseptostomy at 3 days of age for complete transposition of the great arteries with ventricular septal defect and pulmonary stenosis underwent pulmonary root translocation with the lecompte maneuver . this operation has the advantages of maintaining pulmonary valve function , preserving the capacity for growth , and avoiding problems inherent to the right ventricular to pulmonary artery conduit . this patient progressed well for 9 months postoperatively and we report this case of pulmonary root translocation with the lecompte maneuver .

a two - day - old male with whole body cyanosis and tachypnea born at 38 + 2 weeks of gestation with a birth weight of 4,000 g was referred to samsung medical center . his percutaneous oxygen saturation was 60% to 70% , and there was no response to oxygen supply . a grade 3/6 systolic murmur was heard at the left sternal border , and the vital signs were a blood pressure of 70/40 , heart rate of 165 beats per minutes , respiratory rate of 42 breaths per minute , and temperature of 37.2c . echocardiographic findings demonstrated a complete transposition of the great arteries ( tga ) , large peri - membranous ventricular septal defect ( vsd ) , mild pulmonary stenosis ( ps ) with a peak pressure gradient of 23 to 32 mmhg , and a small patent ductus arteriosus ( pda ) . additionally , there was subpulmonic deviation of the outlet septum with a small asymmetric pulmonary valve ( pv ) with fusion . the pv size was 5.86.1 mm ( z - score < 2.5 ) ( fig . after the procedure , the respiration rate stabilized and the percutaneous oxygen saturation was 80% under ambient conditions . the patient was relatively well until five months of age when he started to become increasingly short of breath . eventually , he was admitted for operation , with a body weight of 7.4 kg , and underwent pulmonary root translocation with the lecompte maneuver . after median sternotomy , an inverted y - shaped pericardiostomy was performed . because in situ material was used for the right ventricular outflow tract ( rvot ) reconstruction , the pericardium was not harvested . after pda division , antegrade cold blood cardioplegia was infused through the aortic root cannulation . then , the pulmonary artery and its branches were extensively dissected , from the distal attachments into both pulmonary hila , to be mobilized anterior to right ventriculotomy . the coronary artery pattern was [ 1 ad ; 2 r , cx ] . because of the prominent left anterior descending ( lad ) coronary artery , we planned a lecompte maneuver . without this maneuver , after transection of the proximal ascending aorta , the pulmonary artery root was excised below the pv annulus from the left ventricle ( lv ) ( figs . 2a , 3a ) . the main pulmonary artery was moved anteriorly with the lecompte maneuver . 3b ) , the vsd position was confirmed via the right atrium and right ventriculotomy . a portion of the right ventricular muscle was resected at the conal septum to relieve the lv exit to the aorta . then , the defect in the pulmonary root was closed with a bovine pericardial patch and 6.0 polypropylene running suture . after an end - to - end anastomosis of the transected aorta , the vsd was closed with a bovine pericardial patch and 6.0 polypropylene running suture to create the lv - to - aorta tunnel ( fig . . formation of lv tunneling with this patch during the lv tunneling , we tried to make the patch straightened for the preservation of rv volume . the pv was a functional bicuspid valve with fusion , and the pv annulus size which was measured intraoperatively using hegar dilator was 9 mm . appropriate pv annulus size according to the patient s body surface area ( z - score ) was 10.5 mm , so we performed commissurotomy . the rvot was reconstructed with an excised pulmonary artery root and an in situ autologous pericardial patch that had growth potential on the anterior side ( figs . after the operation , the patient was extubated on the 3rd post - operative day and discharged with no early post - operative complications on the 8th postoperative day . echocardiographic findings 7 days after the operation showed minimal ps ( mean pg=13 mmhg ) with a pv size of 6.8 mm ( z - score < 2.5 ) , and those 6 months after the operation showed mild ps ( mean pg=22 mmhg ) with a mature pv size of 15.8 mm ( z - score=+1.70 ) ( fig . residual arterial septal defect , vsd , or insufficiency of the left ventricular outflow tract were not exhibited . at this time the patient is doing well 9 months postoperatively and has a new york heart association grade of class i. the rastelli procedure was first performed in 1968 and soon became a standard surgical technique for patients with tga associated with a vsd and ps . in addition to excellent relief of ps , the rastelli procedure allows for anatomic correction and achieves good anatomic and physiologic results in the short term . however , late results of this procedure have critical limitations : low long - term survival and an inevitable reoperation on the obstructed conduit . these problems come from the inability of the conduit to grow and from lvot stenosis [ 57 ] . to overcome the limitations of this procedure , pulmonary root translocation ( prt ) was introduced by da silva et al . . with the prt technique , the dissected pulmonary root is implanted in the right ventricle outflow tract to allow for growth of the pulmonary artery . additionally , a more natural lvot is constructed by avoiding making an incision or mobilizing the aorta . long - term follow - up data demonstrated high long - term survival and adequate pulmonary root growth . inspired by da silva , we performed a prt on a five - month - old patient with tga , vsd , and ps . during the operation , great care was taken to avoid injury to the pv and the mitral valve when removing the pulmonary root . unlike da silva et al . , we employed the lecompte maneuver . the translocated pulmonary trunk was close to the sternum , which resulted in an increased risk of bleeding during re - intervention . additionally , we used an in situ autologous pericardium patch during the rvot reconstruction because of the expected growth potential . however , a major drawback is the possible formation of aneurysmal dilatation of the pericardial hood . in this case , questions regarding adequate pv size for pulmonary root translocation have arisen . however , larger numbers of patients and longer follow - up data will be needed to address this point . pulmonary root translocation with the lecompte maneuver can be a candidate procedure with the aim of preserving the growth potential of the native pv .

dendritic cells ( dcs ) are sentinels of the immune system and represent a heterogeneous cell population . the existence of distinct dc subsets is due to their inherent plasticity and to the changing microenvironment modulating their immunological properties . numerous signalling pathways have impacts on dcs . it appears that besides cytokines / chemokines , lipid mediators also have profound effects on the immunogenicity of dcs . some of these lipid mediators exert an effect through nuclear hormone receptors . interestingly , more recent findings suggest that dcs are able to convert precursors to active hormones , ligands for nuclear receptors . some of these dc - derived lipids , in particular retinoic acid ( ra ) , have a central function in shaping t - cell development and effector functions . in this review , we summarize and highlight the function of a set of nuclear receptors ( ppar , ra receptor , vitamin d receptor and glucocorticoid receptor ) in dc biology . defining the contribution of nuclear hormone receptor signalling in dcs can help one to understand the regulatory logic of lipid signalling and allow the exploitation of their potential for therapeutic intervention in various immunological diseases .

traditionally , macrophages and b cells were thought to be the only cell types able to present antigens to t cells and thus elicit immune response . the pioneering work of steinman and cohn showed that a third kind of antigen - presenting cell types , dendritic cells ( dcs ) , are indispensable for the initiation of the adaptive immune response ( steinman and cohn , 1973 ; banchereau and steinman , 1998 ) . the migratory , tissue - resident dcs work at the interface of peripheral tissues and lymphoid organs . these cells are sentinels of the immune system , and they sense and translate environmental cues by sampling and processing extracellular and intracellular antigens ( figure 1 ) . dcs are able to pick up antigens using various uptake mechanisms ( norbury , 2006 ; savina and amigorena , 2007 ) . a key defining feature of this cell type is that after antigen uptake and processing , dcs migrate to lymph nodes , where they present antigens and stimulate t cells ( figure 1 , insert ) . dcs are armed with numerous receptors that detect danger ' signals in the surrounding environment . these signals , associated with an ongoing infection , such as pathogen - associated molecular patterns or pro - inflammatory cytokines , cause dcs to undergo phenotypic changes ( maturation , activation ) that maximize their ability to elicit proliferation of t cells . mature dcs have an extraordinary capacity to activate naive t cells owing to the high expression of various co - stimulatory molecules ( ni and o'neill , 1997 ) . however , besides eliciting immune response , dcs could also provoke immunological tolerance by inducing deletion or anergy ; thus , dcs contribute to limiting autoimmunity ( cools et al , 2007 ) . there are several mechanisms for the maintenance of peripheral tolerance , one of which is when external stimuli reprogramme dcs towards a less activated tolerogenic cell type . dcs form a heterogeneous cell population , which could be classified as plasmacytoid or conventional dcs ( shortman and naik , 2007 ) . dcs could also be subdivided into migratory dcs , which reside in peripheral tissues , or lymphoid organ - resident dcs , which constitute 50% of the lymph node dcs , and all the splenic and thymic dcs ( villadangos and schnorrer , 2007 ) . the molecular details of the regulation of dc differentiation are still poorly characterized , although it was described and suggested that several cytokines and transcription factors are necessary for dc development ( zenke and hieronymus , 2006 ; wu and liu , 2007 ) . flt3 ligand is critical for the development of both conventional and plasmacytoid dc differentiation ; in contrast , gm - csf promotes only conventional dc development ( wu and liu , 2007 ) . in addition , in humans , treatment with il-4 and gm - csf cytokine is used for the ex vivo generation of monocyte - derived dcs ( sallusto and lanzavecchia , 1994 ) . much less is known about the transcriptional regulation of dc development , and only a few putative master ' transcription factors have been identified so far ( zenke and hieronymus , 2006 ) . a microarray study indicated that some nuclear proteins / transcription factors , including ppar and lxr nuclear hormone receptors , are induced during monocyte - derived dc development ( le naour et al , 2001 ) . our global gene expression profiling also showed that 20 out of 48 nuclear receptors are present in human monocyte - derived dcs ( l nagy et al , unpublished results ) . these findings indicate that nuclear receptors are likely to have functions in the differentiation and function of this cell type . nuclear hormone receptors are ligand - activated transcription factors that modulate gene expression through binding to specific hormone response elements . it is well established that ligands / hormones ( i.e. retinoids ( metabolites of vitamin a ) , vitamin d , glucocorticoids ( gcs ) ) of nuclear receptors , besides regulating development and metabolism , also have an impact on the immune system . now this picture is being altered by findings that activators of these receptors can also modulate dc differentiation and function . in the first part of this review , we will provide an introduction to the function of lipids in dc biology , and then summarize the potential function of nuclear hormone receptors in dc differentiation and function . we will focus on rxr heterodimeric receptors ( ppar , rar , lxr and vitamin d receptor ( vdr ) ) and also discuss the potential function of gc receptors in dc function . in the second part , we will provide an overview on how endogenous ligand production for these receptors is taking place in dcs . in the body , there are several tissue compartments in which dcs are likely to be exposed to large amounts of lipids . an obvious one is the gut - associated lymphoid tissue ( galt ) where diet - derived lipids , fatty acids , retinoids and cholesterol are abundant ( figure 1 ) . in the small intestine , the lamina propria is important to maintain peripheral tolerance towards commensal bacterial flora ( nagler - anderson and shi , 2001 ) , and the dietary lipid mediators might contribute to this type of tolerance . it is well established that the atherosclerotic plaques contain macrophages and t cells ; however , dcs were also detected in the plaques ( bobryshev and lord , 1995 ; angeli et al , 2004 ) . in this microenvironment in addition , several other active lipid mediators might be present , which could modulate the migratory and immunological properties of the cells ( figure 1 ) . tissue - resident dcs sense and translate environmental cues by sampling and processing protein antigens ; however , these cells can also present lipid / glycolipid antigens through cd1 cell surface molecules ( brigl and brenner , 2004 ) . cd1s molecules are critical for the presentation of various bacterial glycolipid / peptidolipid antigens ( willcox et al , 2007 ) , but the ways in which endogenous lipids are recognized and presented are still poorly characterized ( tsuji , 2006 ) . an interesting aspect of lipid antigen presentation is that extracellular lipid particles facilitate the uptake of pathogen - derived cd1 ligands . for example , apoptotic bodies from mycobacterium - infected macrophages are efficient vehicles for lipid uptake of uninfected dcs ( schaible et al , 2003 ) ; in addition , a cd1d ligand precursor is delivered to dcs by apolipoproteins ( van den elzen et al , 2005 ) . these results underscore the notion that the local lipid environment has the ability to modify the lipid presentation capacity of dcs . in addition , lipid - derived mediators also elicit intracellular signalling , which alters the maturation and immunogenicity of the antigen - presenting cells . human monocyte - derived dc maturation is induced by oxldl exposure and this effect is mediated by lysophosphatidyl choline through the activation of a g - protein - coupled receptor ( coutant et al , 2002 ) . dcs also sense and integrate lipid signals through pg receptors ; for example , dc migration and cytokine production could be modulated by pge2 and ltb4 . pgd is also an important modulator of dc function ( harizi and gualde , 2005 ) . as we have briefly highlighted , dcs sense the lipid environment by various cell membrane receptors , and now there is an increasing amount of evidence to suggest that dcs could also survey the lipid environment by lipid sensing nuclear hormone receptors . in the following section , we summarize the potential function of these lipid - activated nuclear receptors in dcs . . there are three isotypes of ppars , , and /. these receptors have key physiological functions ; for example , ppar promotes fatty acid oxidation in the liver ( lefebvre et al , 2006 ) , ppar is indispensable for adipocyte differentiation and generally stimulates lipid storage ( willson et al , 2001 ) , and ppar is an important regulator of skeletal muscle lipid oxidation ( barish et al , 2006 ) . recent findings indicated that these receptors , especially ppar , have important functions in dc biology ( figure 2 ) . initially , a comprehensive microarray study indicated that in humans , monocyte - derived dcs have an elevated expression of ppar ( le naour et al , 2001 ) . this finding was consistent with a previous observation indicating that il-4 is a positive regulator of ppar in human and murine monocytes / macrophages ( huang et al , 1999 ) . later , it was confirmed by several laboratories that both the mrna and also the protein of ppar are induced in human monocyte - derived dcs ( gosset et al , 2001 ; nencioni et al , 2002 ; szatmari et al , 2004 ) . in addition , we found that the bona fide ppar target ( fabp4/ap2 ) is highly upregulated upon ppar ligand treatment in developing human dcs . moreover , ex vivo cultured blood - derived conventional ( cd1c+ ) dcs also express ppar. finally , in human lymphoid tissues ( tonsils ) , several ppar and s100 double - positive cells were detected , suggesting that at least a sub - population of lymphoid tissue dcs express this receptor ( szatmari et al , 2004 ) . murine splenic cd11c+ dcs also express ppar ( faveeuw et al , 2000 ) and this nuclear receptor was detected in bone marrow - derived murine dcs ( hammad et al , 2004 ) . the changes in ppar expression during various stages of dc development are still poorly characterized ; however , it was described that the cd1a - negative population of monocyte - derived dcs express more ppar than the cd1a - positive counterparts ( gogolak et al , 2007 ) . the effects of ppar activators on the immunological function and phenotype of ex vivo cultured dcs were investigated in detail . ppar-activated immature dcs have an enhanced phagocytic activity ; in addition , these cells possess a diminished migratory capacity ( angeli et al , 2003 ; szatmari et al , 2004 ; appel et al , 2005 ) . ppar-activated human dcs produce less il-12 and tnf ; moreover , these cells secrete lower amounts of mcp2 , ip-10 and rantes chemokines ( gosset et al , 2001 ; nencioni et al , 2002 ) ( figure 2 ) . furthermore , ppar-instructed dcs have an altered cell surface expression pattern of co - stimulatory molecules ; they express less cd80 but increased cd86 ( gosset et al , 2001 ; nencioni et al , 2002 ; szatmari et al , 2004 ) . these observations appeared to be consistent with a model in which ppar-activated dcs skew the differentiation to a special dc subset that has a reduced th1 activation capacity but an enhanced th2 activation propensity ( faveeuw et al , 2000 ; gosset et al , 2001 ) . others suggested that the activation of the ppar pathway generally reduces / inhibits the immunogenicity of developing human dcs ( nencioni et al , 2002 ; appel et al , 2005 ) . recently , in murine dcs , a similar effect was observed , suggesting that ppar activation has a negative effect on the stimulatory capacity of dcs . more importantly , in the same report , ppar-deficient dcs were used and the authors found that ppar 's effects on the stimulatory capacity of the cells are receptor dependent ( klotz et al , 2007 ) . however , the molecular mechanism of this negative regulation is not clear . by analogy , one suggested mechanism for this activity is the sumoylation of the receptor and recruitment of co - repressors on the promoter region of nf-b - binding sites of various inflammatory genes ( pascual et al , 2005 ) . in dcs , it was suggested that the ppar ligand treatment interfered with the activation of the nf-b and the map kinase pathways ( appel et al , 2005 ) . interestingly , besides these essentially inhibitory activities , ppar appears to possess positive regulatory activities in immune cells as well . our recent global gene expression profiling study revealed that on acute ppar ligand treatment , more than 100 genes were immediately induced and most of these early activated genes fall into the lipid metabolism category ( szatmari et al , 2007 ) . in contrast , immune response - related genes were induced only after 24 h or later , suggesting that ppar activation directly regulates lipid metabolism of these cells but rather indirectly modifies the immune phenotype of dcs . as we have described in the previous section , dcs our results indicated that the ppar ligand coordinately regulates the cd1 gene family expression in human monocyte - derived dcs . ppar-activated dcs express less cd1a ( nencioni et al , 2002 ; szatmari et al , 2004 ) , but have an elevated expression of cd1d . more importantly , the elevated expression of cd1d was coupled to the enhanced capacity to activate a cd1d - dependent cell type , the inkt ( invariant natural killer t ) cells ( szatmari et al , 2004 ) . the lack of inkt cell activation has been implicated in the development of autoimmune conditions , suggesting that inkt cells are intimately linked to sustaining immunological tolerance ( hammond and kronenberg , 2003 ) . we also defined the possible mechanism for this regulation : ppar indirectly stimulated the expression of cd1d through the production of all - trans retinoic acid ( atra ) , the natural ligand of ra receptors ( rars ) ( szatmari et al , 2006a ) . confirming this , it was reported recently that the human cd1d promoter contains a retinoid response element ( chen and ross , 2007 ) . we also described that ppar-activated dcs have an elevated expression of abcg2 , a xenobiotic transporter . these results suggested that the ppar activation modifies the xenobiotic and drug resistance of human dcs ( szatmari et al , 2006b ) . we and others described that dcs express very low levels of ppar. consistent with this finding , ppar ligand treatment has only a marginal effect on monocyte - derived dc differentiation and function ( gosset et al , 2001 ; szatmari et al , 2004 ) . in contrast , it was shown that ppar is present in human epidermal dcs ( langerhans cells ) and that the activation of the ppar pathway blocks the activation and migration of these skin dcs ( dubrac et al , 2007 ) . curiously , dcs also express ppar ( szatmari et al , 2004 ) , but the potential role of these receptors in dc function is not yet characterized . another metabolite receptor , the liver x receptors ( lxrs ) , the sensor of oxidized forms of cholesterol ( zelcer and tontonoz , 2006 ) , were reported to be upregulated during monocyte - derived dc development ; moreover , lxr ligand - treated dcs have a reduced t - cell activation capacity ( geyeregger et al , 2007 ) . the natural ligands of these receptors are poorly characterized ( polyunsatured fatty acids , oxidized fatty acids or cholesterol ) . therefore , much remains to be discovered how the receptor 's endogenous activation takes place . nevertheless , high - affinity synthetic ligands for these receptors are available to probe their function in dcs ex vivo and potentially in vivo . in the next sections , we describe the potential function of two receptors having a high - affinity hormonal ligand in dc biology . activators of gc and vdrs have profound immunosuppressive effects . it was assumed that lymphocytes are the main target of these compounds . however , several in vitro and some in vivo observations suggested that gcs and vitamin d also diminished the immunogenicity of dcs and that these effects might contribute to the impaired immune response and to the anti - inflammatory effects . there are a few recent reviews that provide an overview on the immunosuppressive effects of gcs and vitamin d on dcs ( abe and thomson , 2003 ; hackstein and thomson , 2004 ; van etten and mathieu , 2005 ) , and thus we only briefly discuss the effects of these ligands on dc biology . the immunosuppressive effects of gcs have been well documented and several studies have suggested that , among other cell types , dcs are also affected . gcs decrease the co - stimulatory molecule expression in murine dcs , and therefore these cells have a poor t - cell stimulatory capacity in vitro and in vivo ( moser et al , 1995 ) . in humans , gc - treated monocyte - derived dcs have a reduced t - cell activation capacity ; moreover , gcs interfere with the differentiation of dcs from monocytes ( piemonti et al , 1999 ; rea et al , 2000 ) ( figure 2 ) . the anti - inflammatory effects of gcs are well characterized , but the molecular mechanisms are still poorly defined ( hackstein and thomson , 2004 ) . it has been shown that gcs regulate the immune function of t cells and dcs parallelly by the coordinated activation of the gitr ligand in t cells and the induction of gitr receptors in plasmacytoid dcs . this signalling in dcs leads to a non - canonical nf-b - dependent induction of indolamine 2,3-dioxygenase ( ido ) , a key enzyme that catalyses the initial and rate - limiting step in the degradation of tryptophan and a negative modulator of lymphocyte proliferation ( grohmann et al , 2007 ) . in addition , gcs exert an ido - dependent protection in a model of allergic airway inflammation . the active form of vitamin d also has immunosuppressive effects , and numerous studies have shown that the regulation of dc immunofunctions is an important part of this profound suppressive activity . 1,25 dihydroxy vitamin d3 highly suppresses the activation / maturation of dcs ; moreover , these cells have diminished t - cell activation capacity ( penna and adorini , 2000 ; piemonti et al , 2000 ) ( figure 2 ) . an in vivo murine model has also confirmed that the administration of vdr agonist negatively modulates the stimulatory capacity of dcs . consistent with this finding , vdr - deficient mice have hypertrophy of subcutaneous lymph nodes and an elevated number of mature dcs ( griffin et al , 2001 ) . the ways in which vdr carries out its functions , whether it interferes with positive signalling or induces suppressor molecules directly or simply inhibits dc differentiation and/or maturation is not clear . it was suggested that vdr transcriptionally represses the expression of one of the components of nf-b ( relb ) , thus blocking the activation of dcs ( dong et al , 2003 ) ( figure 2 ) . the extent of this mechanism is not known . in summary , the data obtained to date suggest that the activation of gc or vdr has profound inhibitory or immunosuppressive / tolerogenic effects in dcs ' immunophenotype and that these pathways are potentially amenable to therapeutic exploitation . in the next section , we discuss the function of retinoids , which show a much more complex behaviour having both repressive and activator functions in dcs . it is well known that retinoids ( derivatives of vitamin a and ligands of rar and rxr receptors ) exert a modulatory effect on the immune system . vitamin a deficiency causes immune dysfunction and increases the susceptibility of an individual to infectious diseases , thus contributing to elevated child morbidity and mortality ( underwood and arthur , 1996 ) . it was reported that mouse splenic dcs are less stimulatory ( bedford and knight , 1989 ) on ra treatment ; however , retinoid - treated langerhans cells have an enhanced t - cell activation capacity ( meunier et al , 1994 ) . moreover , ra promotes the differentiation and maturation of monocytes to dc - like cells ( mohty et al , 2003 ) and these compounds enhance the dna - binding activity of nk-b , thus promoting the maturation of dcs ( geissmann et al , 2003 ) ( figure 2 ) . it should be noted that retinoids also provoke apoptosis in developing dcs through rarrxr heterodimers ( geissmann et al , 2003 ) . it was also described that ra pretreated dcs , if injected into tumours in mice , showed an increased accumulation in draining lymph nodes . it was concluded that the enhanced dc migration was due to the elevated matrix metalloproteinase production and the concurrently diminished expression of inhibitors of metalloproteinase ( darmanin et al , 2007 ) . moreover , ra - treated monocyte - derived dcs produce an elevated level of tgf and il-6 and these ra - instructed dcs acquire several attributes characteristic of mucosal dcs ( saurer et al , 2007 ) . our laboratory also investigated the effects of activation of the rars using synthetic retinoids on human monocyte - derived dc differentiation and found that these cells have an enhanced inkt cell activation capacity as a consequence of the induction of cd1d ( szatmari et al , 2006a ) ; moreover , our gene expression profiling indicates that a large number of genes are regulated by retinoids leading to the activation of multiple pathways . all - trans ra is an agonist of rar ; however , an isomeric form , 9-cis - ra , can activate both rar and rxr ( mangelsdorf and evans , 1995 ) ; therefore , 9-cis - ra has pleiotropic effects and can modulate the activity of several nuclear receptors in principle . interestingly , 9-cis - ra exerts a suppressive effect on human monocyte - derived dc differentiation , probably through the activation of the pparrxr heterodimer ( zapata - gonzalez et al , 2007 ) . we also tested the effects of a synthetic and specific activator of rxr ( lg268 ) on dc differentiation and obtained complex phenotypic changes , suggesting that probably multiple nuclear receptor heterodimers are affected ( i szatmari and l nagy , unpublished results ) . exposure to retinoids has a complex phenotype in dcs , and as we will demonstrate in the next section , ra has a profound effect on lymphocyte homing and activation as well . in this review , we have systematically analysed the potential function of the activation of various nuclear receptors in dc development and function . it should be emphasized that most studies used high - affinity synthetic ligands for the activation of the nuclear hormone receptors and for probing their biological functions . the other is whether dcs are actively participating in the production of nuclear hormone receptor ligands or simply get those through endocrine or paracrine mechanisms . it should be noted that in the serum , a large number of compounds are present as biologically inactive precursors . however , on activation ( oxidation / hydroxylation ) , the inactive compounds can be converted to the active forms . for example , vitamin d3 must be hydroxylated to 1,25 dihydroxy vitamin d3 , which is a high - affinity ligand of vdr ( okuda et al , 1995 ) , and vitamin a ( retinol ) first gets oxidized to retinaldehyde ; thereafter , this compound is converted to ra , which is an agonist of rars ( duester , 2000 ) . similarly , cortisone is converted to cortizole , a ligand of gr ( seckl and walker , 2001 ) ( figure 3 ) . additionally , hydroxylation of cholesterol at the position of 27 leads to 27-hydroxy - cholesterol , a potent activator of the lxr receptors ( fu et al , 2001 ) , and finally polyunsaturated fatty acids are oxidized by lipoxygenases and these oxidized compounds are relatively potent ligands of the ppar receptor ( nagy et al , 1998 ; huang et al , 1999 ) . the organic and cellular localization of these steps and the enzymes that catalyse these reactions are well characterized for some cell types . most of these chemical transformations occur in the liver and the kidney but , significantly , some of these steps can also be found in dcs . the most studied nuclear receptor ligand in the context of dcs is ra , the active form of vitamin a. in a breakthrough study , iwata et al ( 2004 ) discovered that murine intestinal dcs produce and release ra , and this dc - derived ra instructs the gut - homing tropism of t cells by the induction of the expression of 47 integrin and ccr9 receptors . this study established that a dc - derived nuclear receptor ligand is able to reprogramme the local t cells and imprint t - cell tropism . interestingly , several recent publications suggest that ra , in combination with other immunological mediators , modulates the differentiation of various lymphocyte populations in the galt . in the intestine , ra blocks the il-6- and tgf-driven induction of the pro - inflammatory il-17-producing t ( th17 ) cells , but promotes the differentiation of the tolerogenic regulatory t ( treg ) cells ( mucida et al , 2007 ) . these findings were confirmed by others : intestinal dc - produced ra enhances the tgf-dependent conversion of peripheral t cells to foxp3-positive treg cells , suggesting that ra works as a mediator of gut / oral tolerance in vivo ( benson et al , 2007 ; coombes et al , 2007 ; sun et al , 2007 ) . gut - associated dcs also promote b cell gut tropism by the upregulation of the aforementioned receptors ; additionally , they enhance the il-6-dependent iga secretion of b cells ( mora et al , 2006 ) . collectively , these findings underscore that dc - derived ra has a prominent function in the regulation of gut - associated immune processes . it should be mentioned that ra itself could induce the expression of 47 and ccr9 on t cells and thus imprint gut tropism , but for the study of the other effects of ra on lymphocytes , in most cases dc - t cell co - cultures were used . therefore , formally , it is possible that ra also affects the immunophenotype of the dcs and that these reprogrammed ' dcs promote treg development or b - cell iga production . another interesting point is to address which factors regulate ra production and which enzymes are responsible for the unique capacity of the intestinal dcs to produce ra . there is a remarkable selectivity of this phenotype . in vivo , in contrast , non - gut lymphoid tissue dcs are unable to generate ra ( iwata et al , 2004 ) . intestinal dcs express several enzymes that might participate in ra synthesis , the most important ones being the retinaldehyde dehydrogenases ( raldhs ) , which catalyse the retinaldehyde oxidation to ra ( figure 2 ) . dcs of peyer 's patches mostly express raldh1 ( aldh1a1 ) ; in contrast , mesenteric lymph node dcs mostly express raldh2 ( aldh1a2 ) ( iwata et al , 2004 ) . a specialized sub - population of mesenteric lymph node dcs , cd103 + cells , promote the generation of treg cells and these dcs show an elevated expression of raldh2 ( coombes et al , 2007 ) . interestingly , we observed that ppar-instructed human monocyte - derived dcs also produce ra ; moreover , raldh2 and rdh10 ( retinol dehydrogenase 10 ) enzymes were upregulated in these cells ( szatmari et al , 2006a ) . it is well established that raldh2 is indispensable for embryonic ra production ( niederreither et al , 1999 ) . significantly , a recent report suggested that rdh10 might catalyse the first oxidation step of retinol , a critical step for embryonic ra generation ( sandell et al , 2007 ) . further investigations are needed to define what kind of mechanisms regulate ra production in dcs and which enzyme / s is / are rate limiting for ra synthesis . once these issues are clarified , one would be able to define the in vivo contribution of dc - derived ra signalling in the various immunological processes described earlier and also beyond those . dcs are also able to activate other vitamins : the inactive form of vitamin d , 25-hydroxy vitamin d3 , is converted to 1alpha,25 dihydroxy vitamin d3 during the ex vivo differentiation of human dcs . this conversion is catalysed by 25(oh)d3 - 1 alpha hydroxylase , which is present in developing dcs ; in addition , this enzyme is upregulated on maturation of dcs ( fritsche et al , 2003 ; hewison et al , 2003 ) . consistent with these findings , the production of the vdr ligand is claimed to negatively regulate the early differentiation of monocyte - derived dcs . of note , dermal dcs are able to directly convert vitamin d3 to 1,25 dihydroxy vitamin d3 in the skin , and this active form of vitamin d instructed the local t cells to express ccr10 , thus enabling them to migrate to the epidermis ( sigmundsdottir et al , 2007 ) . remarkably , these results suggested that similar to ra , which elicits gut - homing specificity of t cells , the active form of vitamin d imprints skin - homing specificity of t cells ( figure 4 ) . human monocyte - derived dcs are also able to convert the inactive cortisone to the high - affinity gr ligand , cortisole as a result of the upregulation of 11beta hydroxysteroid reductase . in addition , dc differentiation is inhibited by the administration of physiological concentration of cortisone , suggesting that this pathway enhances the gc - dependent negative modulation of dc development ( freeman et al , 2005 ) . finally , as far as the ppar ligand is concerned , we observed that during monocyte - derived dc development the bona fide ppar targeting the fabp4/ap2 gene is upregulated , especially when we culture the cells in human serum instead of fbs ( szatmari et al , 2004 ) . we extended this finding and observed that most of these genes whose expression was upregulated on synthetic ppar ligand treatment also showed an elevated expression if dcs were cultured in human serum - containing medium ( szatmari et al , 2007 ) . these results suggested that in the presence of human serum , the ppar ligands are generated / accumulated in dcs . indeed , a recent paper suggested that lysophosphatidic acid and cardiolipin are abundant components of the human serum and that these lipid species might contribute to the activation of ppar ( leslie et al , 2008 ) . these results underscore the notion that , remarkably , dcs are actively participating in the production of several ligands of nuclear receptors . moreover , some of these ligands are likely to be released and are able to locally modulate the function of other immune cell types . dcs are indispensable for the initiation of primary immune response , but now it is well established that these cells are able to orchestrate the entire immune response , including the regulation of peripheral immune tolerance . therefore , it is of importance to identify and characterize the regulatory processes underpinning these changes . it is becoming increasingly evident that members of the nuclear hormone receptor superfamily are involved in the regulation of dc biology . this group of transcription factors also represents a gateway to manipulating dcs for therapeutic use . in clinical practice , tumour antigen - loaded dcs are intensively investigated tools to elicit antitumour immune response . ex vivo differentiated dcs may be loaded with tumour antigens and injected back to patients , or they can be used for ex vivo expansion of antitumour lymphocytes . improving anticancer immunotherapies appear to largely depend on how dc immunogenicity , survival and migration are regulated , and thus it is very important to find ways to modulate antigen presenting , migratory capacity and viability of dcs ( nencioni et al , 2008 ) . there are several ways to manipulate dcs ex vivo ; administration of cytokine cocktails or other signal molecules can modify the dc function and activation state . as we have demonstrated in this review , natural or synthetic ligands of nuclear hormone receptors can also modulate dc functions . in the future , it will be important to explore and exploit the benefit of ligand treatment to modulate dc immunogenicity in vivo . thus , in patients with an altered lipid profile , these receptors might be overactivated and this can adversely modify the immune phenotype of their dcs . as an example , we have described that serum lipoproteins skewed dc development towards a cd1a dc phenotype , which has a distinct cytokine and chemokine production profile ( gogolak et al , 2007 ) . until now , most of the studies on nuclear receptors used ex vivo dc models and only a few reports investigated tissue - specific nuclear receptor knockout models ( griffin and kumar , 2003 ; klotz et al , 2007 ) . in the future , it will be of great importance to generate dc - specific nuclear receptor deletion models . these models will almost certainly prove to be helpful to define the receptor specificity of the effects of receptor agonists . in addition , these systems will help to define the in vivo function of these receptors in dcs . the in vivo approaches might also clarify some of the controversies that still exist in the field . however , as we have emphasized concerning human immunotherapy , ex vivo dc models are also used . thus , it is important to characterize the in vitro effects of ligands of nuclear hormone receptors , not in small part , because there are major differences between mice and humans regarding dc functions , and murine models might not always be predictive of human responses . dcs are a very heterogeneous cell population ( villadangos and schnorrer , 2007 ) , and until now most data were derived from human monocyte - derived dcs . in the future , it would also be important to define the function of nuclear receptors / ligands on the various subtypes of dcs and dc precursors . and , finally , activators of nuclear receptors are widely used in clinical practice , for example , ligands of the ppar ( rosiglitazone and pioglitazone ) are applied to treat type ii diabetes ( willson et al , 2001 ) ; in addition , gcs are frequently used as immunosuppressive agents . defining the exact functions of these activators in various immune cell types could help in exploiting their potential for therapeutic intervention against various immunological diseases .

background : drug - induced liver injury is a common cause of liver damage and the most frequent reason for withdrawal of a drug in the united states . the symptoms of drug - induced liver damage are extremely diverse , with some patients remaining asymptomatic.methods:this observational study is based on data of arzneimittelsicherheit in der psychiatrie , a multicenter drug surveillance program in german - speaking countries ( austria , germany , and switzerland ) recording severe drug reactions in psychiatric inpatients . of 184234 psychiatric inpatients treated with antidepressants between 1993 and 2011 in 80 psychiatric hospitals , 149 cases of drug - induced liver injury ( 0.08% ) were reported.results:the study revealed that incidence rates of drug - induced liver injury were highest during treatment with mianserine ( 0.36% ) , agomelatine ( 0.33% ) , and clomipramine ( 0.23% ) . the lowest probability of drug - induced liver injury occurred during treatment with selective serotonin reuptake inhibitors ( [ 0.03% ) , especially escitalopram [ 0.01% ] , citalopram [ 0.02% ] , and fluoxetine [ 0.02% ] ) . the most common clinical symptoms were nausea , fatigue , loss of appetite , and abdominal pain . in contrast to previous findings , the dosage at the timepoint when dili occurred was higher in 7 of 9 substances than the median overall dosage . regarding liver enzymes , duloxetine and clomipramine were associated with increased glutamat - pyruvat - transaminase and glutamat - oxalat - transaminase values , while mirtazapine hardly increased enzyme values . by contrast , duloxetine performed best in terms of gamma - glutamyl - transferase values , and trimipramine , clomipramine , and venlafaxine performed worst.conclusions:our findings suggest that selective serotonin reuptake inhibitors are less likely than the other antidepressants , examined in this study , to precipitate drug - induced liver injury , especially in patients with preknown liver dysfunction .

the liver , the central organ of biotransformation , is particularly prone to oral medication - related toxicity due to high concentrations of drugs and their metabolites in portal blood rather than in the actual target area of the central nervous system . it is , however , difficult to attribute liver damage to a specific medication in clinical practice ( meyer , 2000 ) . the susceptibility of an individual to drug - induced liver injury ( dili ) depends on multiple genetic and epigenetic factors , age , gender , weight , and alcohol consumption that influence the occurrence of hepatic adverse effects ( krhenbhl and kaplowitz , 1996 ) . older patients seem more vulnerable , and women have a stronger tendency to toxic liver reaction than men ( meyer , 2000 ) ; ethnic differences have also been reported ( evans , 1986 ) . genetic metabolic variability is the most significant susceptibility factor in drug - induced liver toxicity . enzyme polymorphisms can cause a slowing or complete disruption of enzyme function , which in turn results in the inefficient processing of drugs ( shenfield and gross , 1999 ) . this may not always result in corresponding liver damage but does contribute to an increased toxicity of substances . the majority of drugs and almost all psychotropic drugs are metabolized by the enzyme cyp450 . due to genetically determined polymorphisms of cyp450-isoenzymes , individuals can be categorized as poor , intermediate , extensive , or superextensive metabolizers ( miners and birkett , 1998 ; shenfield and gross , 1999 ; wilkinson , 2004 ) . if a poor metabolizer receives medication containing several substrates or inhibitors of the same isoenzyme , the risk of a toxic reaction increases owing to a slower drug metabolism . as most psychotropic drugs are a substrate of cyp2d6 ( ingelman - sundberg , 2005 ) , this cytochrome is especially significant in the pharmacokinetic interaction . approximately 5% to 10% of caucasians have reduced or nonexistent cyp2d6 activity and are therefore at risk of toxicity when receiving psychotropic treatment ( transon et al . , 1996 ; griese et al . 1998 ; a further important consideration is whether patients with preexisting liver dysfunction have a higher risk of hepatotoxic reactions . although little information from controlled studies exists , there are indications that patients with preexisting liver disorders generally do not display an increased risk of drug - induced hepatotoxicity . it is more likely that preexisting liver damage negatively affects the ability of the liver to regenerate in the case of a hepatotoxic reaction ( chang and schiano , 2007 ) . possible symptoms are tiredness , lack of appetite , nausea , vomiting , fever , a feeling of pressure in the upper right region of the abdomen , joint and muscle pain , pruritus , rashes , and jaundice ; the latter is the only symptom directly indicative of the liver s involvement ( chang and schiano , 2007 ) . to diagnose asymptomatic toxic liver damage early , this involves the measurement of the glutamat - oxalat transaminase ( got ) , glutamat - pyruvat - transaminase ( gpt ) , and gamma - glutamyl - transferase ( -gt ) in serum which , if found to be normal , indicates that there has been no disruption to liver function . got and gpt are also well known as the enzyme aspartate aminotransferase ( ast ) and alanine aminotransferase ( alt ) , respectively . it is important to consider the possibility of dili when prescribing psychotropic drugs and to record a detailed history of all medication taken by the patient , with particular attention paid to the length of use , the dose , and the time between the intake of medication and appearance of symptoms . the latency period involved here can vary between a few days and some months and , as liver damage may result from other causes such as viral , autoimmune , alcohol - induced hepatitis , and acute morbus wilson , the diagnosis of drug - induced toxic liver - damage is often a diagnosis of exclusion ( norris et al . , 2008 ) . ( 2014 ) developed practice guidelines for diagnosing and managing dili . the hepatic pattern of damage can be classified as predominantly hepatocellular , predominantly cholestatic , or a hepatocellular / cholestatic mixture and is important , as these patterns are of varying severity . the drugs also cause drug - specific patterns of liver damage revealing increased values of transaminases ( got and gtp ) and/or cholestasis ( -gt , alkaline phosphatase [ zimmerman , 1999 ; andrade et a. , 2004 ] ) . a slight increase in transaminases or -gt levels to twice the norm without a rise in bilirubin is often of no clinical significance and in spite of continued medication , can simply disappear . this is a phenomenon often observed in antiepileptic or mood - stabilizing therapy ( yatham et al . , 2002 ) . these small functional changes must still be checked and in the case of a further elevation in liver enzyme levels medication must be discontinued ( voican et al . , 2014 ) . the prognosis of dili is generally good , and less severe forms heal quickly and completely ( hayashi and fontana , 2014 ) . it is difficult to obtain figures regarding hepatotoxic drug reactions , as systematic epidemiological analyses are seldom done and observations are not conducted for a long enough period to have any true validity . drug surveillance programs permit an early detection of adverse drug reactions ( adrs ) and this may minimize consequences . the arzneimittelsicherheit in der psychiatrie ( amsp ) study is one such program in the field of psychiatry systematically evaluating severe adrs of psychotropic medication in inpatients . the amsp produces a database of these adrs registered in the participating psychiatric clinics in austria , germany , and switzerland ( for details on amsp methods , see grohmann et al . in the present study , we have used this database to analyze the elevation of liver enzymes with a particular focus on sociodemographic data and the significance of clinical manifestations as well as transaminase levels measured during antidepressant ( ad ) monotherapy and combination therapies . the amsp program aims for a continuous detection of severe adrs resulting from psychotropic treatment . these are evaluated during inpatient treatment . in our study , we analyzed data from 80 university , municipal , or state psychiatric hospitals or departments participating in the amsp program in 1993 to 2011 . information on severe adrs is collected from clinicians on a regular basis by psychiatrists as drug monitors who use a standardized questionnaire to document cases . the drug monitors get in touch with ward psychiatrists at regular intervals and severe adverse drug reactions are reported at weekly meetings of the medical staff ( grohmann et al . , 2004 ) . information is collected on the details of adverse events as well as on patient demographics and nonpsychotropic drug intake . it includes alternative hypotheses on the causes of the adr , relevant risk factors , measures undertaken , and previous exposure to the drug . senior doctors of each hospital involved review the cases that are later discussed at central and regional case conferences , which take place 3 times per year . participants comprise hospital drug monitors , representatives from the national authorities regulating drugs , and drug safety experts from the pharmaceutical industry . following discussions and analyses , adr probability ratings are assigned and sent to the relevant authorities , and pharmaceutical companies receive the case questionnaires , which are also stored in the amsp central database . based on the amsp study guidelines ( grohmann et al . , 2004 ) and recommendations of hurwitz and wade ( 1969 ) and seidl et al . the adr probability rating system defines the following grades of probability beginning with grade1 , in which adr is possible , that is , the risk of adr is not known or the probability of another cause other than the drug in question is > 50% . grade 2 is defined as probable , with a known reaction , time course , and dosage for a specific drug . grade 3 is categorized as definite , meaning a reexposure to the drug again causes the adr . grade 4 signifies questionable or not sufficiently documented . in cases where an adr results from a pharmacodynamic interaction of 2 or more drugs , each drug is given a rating of possible , probable , or definite according to the given facts . furthermore , drug - use data are collected twice per year from all hospitals participating in the amsp program ; the number of all inpatients and the mean treatment duration of all patients per year are also recorded . documentation of adrs occurs when the value for one of the liver enzymes ( got / ast , gpt / alt , -gt , or alkaline phosphatase ) exceed 5 times the upper normal values ( severe as defined by the amsp , based on the judgment of hepatologic experts ) or when there are severe clinical symptoms and/or cholestasis . the threshold of 5 times the upper limit of normal got and gpt values have been proposed in the literature to avoid unnecessary withdrawal of substances ( aithal et al . , 2011 ) . maximal levels of each liver enzyme are recorded in the amsp in all dili cases ; mean maximum values per drug were evaluated for this analysis . only drugs prescribed more than 2000 times within the overall study population were included in the analyses . our retrospective analysis employs data extracted from the anonymized databank of the amsp drawn from all 80 participating hospitals between 1993 and 2011 . detailed information on the hospitals participating in the program can be found online ( www.amsp.de ) . the informed consent of participants was not required , as the data analyzed were derived from an anonymized databank . the amsp drug surveillance program was approved by the leading boards of each participating institute prior to implementation , and the ethics committee of the university of munich formally approved evaluations based on the amsp databank . incidence rates of hepatotoxicity were calculated as the percentage of inpatients receiving a specific ad or ad subclass and presented together with their 95% cis . regarding the low actual number of cases and the significant number of inpatients involved , the ci was calculated employing the exact method rather than one of the approximate methods ( vollset , 1993 ) . the statistical program r was used to generate the figures ( r core team , 2014 ) . incidence rates of hepatotoxicity were calculated as the percentage of inpatients receiving a specific ad or ad subclass and presented together with their 95% cis . regarding the low actual number of cases and the significant number of inpatients involved , the ci was calculated employing the exact method rather than one of the approximate methods ( vollset , 1993 ) . the statistical program r was used to generate the figures ( r core team , 2014 ) . a total of 184234 inpatients were treated with antidepressants . in 147 inpatients ( and 149 cases , as 2 inpatients suffered from dili twice ) a severe hepatic adr was observed ( 0.08% ) . within 27 of 149 cases only ads were imputed with the remaining inpatients suffering toxicity from an ad in combination with other psychotropic drugs . the majority of all monitored inpatients treated with antidepressants ( 56.5% ) were suffering from depression . inpatients under surveillance were predominantly female ( 63.1% ) . a total 75.2% of inpatients suffering from dili were diagnosed with depression , followed by 9.4% with the diagnosis of schizophrenia ( table 1 ) . thus , dili patients differed significantly in their diagnostic distribution from the total ad population . age and sex distribution , on the other hand , did not differ in dili patients from all monitored ad patients . age , sex , and international classification of diseases version 10 ( icd-10 ) diagnosis of patients monitored during the period of 19932011 suffering from dili due to ads and the total population under surveillance ( 149 cases of dili ) abbreviations : dili , drug - induced liver injury ; ad , antidepressant ; n , number . in 147 inpatients ( and 149 cases ) , dili frequencies for the different single substances as well as classes of ads are given in table 2 and figures 1 and 2 . incidence of dili and median dosages among drug classes ( n=149 cases of dili and 184.234 patients monitored overall , respectively ) * other tcas : amitryptilinoxid , desipramine , dibenzepin , imipramine . * * no case of milnacipran and tianeptin ; multiple nominations possible . drug - induced liver injury ( dili ) per antidepressant ( ad ) classes / subgroups in percent of exposed patients , only cases where ad subgroups were imputed alone for dili , and only substance classes imputed 3 times or more ( except agomelatine due to its delayed implementation , which was imputed 2 times ) . drug - induced liver injury ( dili ) per antidepressant ( ad)/single substance in percent of exposed patients , only cases where single ads were imputed alone , and just substance classes imputed 3 times or more were included ( except agomelatine due to its delayed implementation , which was imputed 2 times ) . as for ad classes , the subgroup of tricyclic and tetracyclic ads showed the most unfavorable profiles in terms of dili , while the subgroup of serotonin reuptake inhibitors ( ssris ) had the lowest rates of dili ( all cases as well as ssris alone cases ) . as for single drugs , mianserine , agomelatine , and clomipramine showed the highest frequencies of dili with 0.36% , 0.33% , and 0.23% , respectively . trazodone ( the only serotonin antagonist and reuptake inhibitor ) , serotonin norepinephrine reuptake inhibitors ( snris ) , and noradrenergic and specific serotonergic antidepressant ( nassa ) obtained similar results in between . mianserine was added to the tricyclic and tetracyclic ads according to existing literature ( benkert et al . , nevertheless , this can be argued , as some authors add it towards the nassa group due to its similar chemical structure . in 104 of 149 cases , ads were imputed to be solely responsible for dili , 96 cases were registered where only one ad was imputed , and 8 cases where 2 ads or more were imputed in combination . the drugs listed as other tricyclic antidepressants ( 9 cases of dili ) were amitriptylinoxid ( 1 case ) , desipramine ( 1 case ) , dibenzepine ( 6 cases ) , and imipramine ( 1 case ) . the substances mentioned as other ads were nefazodone ( 1 case ) and bupropion ( 1 case ) . the group of monoaminooxidase ( mao ) inhibitors consisted of tranylcypromine ( 3 cases ) and moclobemide ( no case ) . the substances metioned in other tcas ( tricyclic antidepressants ) , other ads , and mao inhibitors were prescribed < 2000 times ; hence , these single drugs were not included in the analyses of the present study . an exception was made for agomelatine , due to the particular interest in this drugs hepatotoxicity . the results of agomelatine , however , have to be interpreted with caution , as it was not introduced until april 2009 . therefore , the observation period for agomelatine was significantly shorter than for all other drugs observed since 1993 . as presented in table 2 , there were differences in the median dosages between the drugs deemed responsible for dili and those for all monitored inpatients treated with ads . within the ssri subgroup , escitalopram , citalopram , and sertraline were prescribed at double the dosage compared with all monitored inpatients at the time when dili appeared . also within the snri , noradrenalin reuptake inhibitor , and nassa subgroups , higher dosages compared with the median dosage for all patients monitored only maprotiline was prescribed at a lower dosage at the moment of dili , while all the other substances of this subgroup were prescribed at higher dosages in cases of dili . the most prevalent drug class combination was the one of ads and antipsychotic drugs ( aps ) , in 31 cases within our study . first , olanzapine was implicated in dili ( 6 cases ) , followed by clozapine ( 3 cases ) , and other aps held responsible for dili in only 1 to 2 cases ( haloperidol , melperone , chlorprothixene , quetiapine , perazine , levomepromazine , promethazine , and risperidone ) . valproic acid was responsible for 3 dili cases followed by carbamazepine , galantamine , pregabaline , and lamotrigine , implicated in only one case each . maximum gamma - gt and transaminase ( glutamat - oxalat - transaminase [ got ] and glutamat - pyruvat - transaminase [ gpt ] ) values per dili case were evaluated for the time period from 2003 to 2011 ( values for agomelatine from 2009 to 2011 , as agomelatine was introduced in 2009 ) . as there are small deviations in terms of maximum got ( also known as aspartate - aminotransferase or ast ) , gpt ( also known as alanin - aminotranferase or alt ) , and alkaline phosphatase values across the participating institutions , a 5-fold increase in enzyme values was determined as dili . from 2003 on , measurement of liver enzymes was done at all participating hospitals at a temperature of 37c . prior to this , measurement was done at 15 to 20c , resulting in lower values for varying time periods at the different hospitals . duloxetine , clomipramine , and paroxetine were mainly responsible for high gpt values , while mirtazapine affected gpt values least . in terms of got values , duloxetine and clomipramine performed worst , and again mirtazapine had the least influence on got values . regarding -gt , duloxetine performed best , while trimipramine , clomipramine , as well as venlafaxine increased -gt values most ( figure 3a - c ) . the duration of treatment when dili occurred was different among the antidepressants ; mianserine was taken for 22 days on average , while mirtazapine was taken for 40 days . ( a ) gamma - glutamyl - transferase ( gamma - gt ) mean maximum values of single substances ( imputed alone for a minimum of 3 times except agomelatine due to its delayed implementation , which was imputed 2 times ) . ( b ) glutamat - oxalat - transaminase ( got ; also known as aspartate - aminotransferase [ ast ] ) mean maximum values of single substances ( imputed alone for a minimum of 3 cases , except agomelatine due to its delayed implementation , which was imputed 2 times ) . ( c ) glutamat - pyruvat - transaminase ( gpt ; also known as alanin - aminotransferase [ alt ] ) mean maximum values of single substances ( imputed alone for a minimum of 3 cases except agomelatine due to its delayed implementation ) . for inpatients with no preexisting liver damage , the mean maximum values for -gt , got , and gpt were 240 , 202 , and 285u / l , respectively , when dili was diagnosed . cases with preknown liver damage presented maximum -gt , got , and gpt mean values of 525 , 402 , and this indicates that preknown liver damage inpatients had more than doubled mean maximum values for -gt transaminases than subjects with normal liver status at the time when dili appeared . in our study sample , preexisting hepatic injury was the most common risk factor by far ( 59 cases ) , followed by substance abuse , mostly alcohol ( 20 cases ) . the most common clinical symptoms were nausea , fatigue , loss of appetite , and abdominal pain . a total of 27 inpatients showed clinical symptoms , while the majority did not show any . in 8 cases , the ad treatment remained and dosage was reduced , while in all other cases the drug was withdrawn after dili was assessed . within 55 cases , dili disappeared totally , while in 85 cases dili improved . within 9 cases only one case of acute liver failure occurred in a 20-year - old woman with a predamaged liver resulting from an overdose of paracetamol . at the time of admission to the psychiatric ward , the transaminase values were normal . she had been on a medication of 150 mg doxepine ( for 3 days ) and 10 mg olanzapine ( for 6 days ) . the patient s liver enzymes increased rapidly , and clinical symptoms such as vomiting , nausea , and epigastric pain set in . in the following laboratory analysis , a hepato - toxicity was identified ( bilirubin 3.8mg / dl , gpt 8827u / l , got 7363u / l , lactate dehydrogenase as soon as acute liver failure was diagnosed , the patient was transferred to the intensive care ward where she was under the care of the transplantation consulting team . the hepatotoxic effects of doxepine and olanzapine have been discribed in previous literature , but to our knowledge such a severe case has not been presented so far . a total of 184234 inpatients were treated with antidepressants . in 147 inpatients ( and 149 cases , as 2 inpatients suffered from dili twice ) a severe hepatic adr was observed ( 0.08% ) . within 27 of 149 cases only ads were imputed with the remaining inpatients suffering toxicity from an ad in combination with other psychotropic drugs . the majority of all monitored inpatients treated with antidepressants ( 56.5% ) were suffering from depression . inpatients under surveillance were predominantly female ( 63.1% ) . a total 75.2% of inpatients suffering from dili were diagnosed with depression , followed by 9.4% with the diagnosis of schizophrenia ( table 1 ) . thus , dili patients differed significantly in their diagnostic distribution from the total ad population . age and sex distribution , on the other hand , did not differ in dili patients from all monitored ad patients . age , sex , and international classification of diseases version 10 ( icd-10 ) diagnosis of patients monitored during the period of 19932011 suffering from dili due to ads and the total population under surveillance ( 149 cases of dili ) abbreviations : dili , drug - induced liver injury ; ad , antidepressant ; n , number . in 147 inpatients ( and 149 cases ) , 19 single substances were solely held responsible for dili . in all other cases , dili frequencies for the different single substances as well as classes of ads are given in table 2 and figures 1 and 2 . incidence of dili and median dosages among drug classes ( n=149 cases of dili and 184.234 patients monitored overall , respectively ) * other tcas : amitryptilinoxid , desipramine , dibenzepin , imipramine . * * no case of milnacipran and tianeptin ; multiple nominations possible . drug - induced liver injury ( dili ) per antidepressant ( ad ) classes / subgroups in percent of exposed patients , only cases where ad subgroups were imputed alone for dili , and only substance classes imputed 3 times or more ( except agomelatine due to its delayed implementation , which was imputed 2 times ) . drug - induced liver injury ( dili ) per antidepressant ( ad)/single substance in percent of exposed patients , only cases where single ads were imputed alone , and just substance classes imputed 3 times or more were included ( except agomelatine due to its delayed implementation , which was imputed 2 times ) . as for ad classes , the subgroup of tricyclic and tetracyclic ads showed the most unfavorable profiles in terms of dili , while the subgroup of serotonin reuptake inhibitors ( ssris ) had the lowest rates of dili ( all cases as well as ssris alone cases ) . as for single drugs , mianserine , agomelatine , and clomipramine showed the highest frequencies of dili with 0.36% , 0.33% , and 0.23% , respectively . trazodone ( the only serotonin antagonist and reuptake inhibitor ) , serotonin norepinephrine reuptake inhibitors ( snris ) , and noradrenergic and specific serotonergic antidepressant ( nassa ) obtained similar results in between . mianserine was added to the tricyclic and tetracyclic ads according to existing literature ( benkert et al . , nevertheless , this can be argued , as some authors add it towards the nassa group due to its similar chemical structure . in 104 of 149 cases , ads were imputed to be solely responsible for dili , 96 cases were registered where only one ad was imputed , and 8 cases where 2 ads or more were imputed in combination . the drugs listed as other tricyclic antidepressants ( 9 cases of dili ) were amitriptylinoxid ( 1 case ) , desipramine ( 1 case ) , dibenzepine ( 6 cases ) , and imipramine ( 1 case ) . the substances mentioned as other ads were nefazodone ( 1 case ) and bupropion ( 1 case ) . the group of monoaminooxidase ( mao ) inhibitors consisted of tranylcypromine ( 3 cases ) and moclobemide ( no case ) . the substances metioned in other tcas ( tricyclic antidepressants ) , other ads , and mao inhibitors were prescribed < 2000 times ; hence , these single drugs were not included in the analyses of the present study . an exception was made for agomelatine , due to the particular interest in this drugs hepatotoxicity . the results of agomelatine , however , have to be interpreted with caution , as it was not introduced until april 2009 . therefore , the observation period for agomelatine was significantly shorter than for all other drugs observed since 1993 . as presented in table 2 , there were differences in the median dosages between the drugs deemed responsible for dili and those for all monitored inpatients treated with ads . within the ssri subgroup , escitalopram , citalopram , and sertraline were prescribed at double the dosage compared with all monitored inpatients at the time when dili appeared . also within the snri , noradrenalin reuptake inhibitor , and nassa subgroups , higher dosages compared with the median dosage for all patients monitored were observed when dili occurred . within the tricyclic and tetracyclic class , only maprotiline was prescribed at a lower dosage at the moment of dili , while all the other substances of this subgroup were prescribed at higher dosages in cases of dili . the most prevalent drug class combination was the one of ads and antipsychotic drugs ( aps ) , in 31 cases within our study . first , olanzapine was implicated in dili ( 6 cases ) , followed by clozapine ( 3 cases ) , and other aps held responsible for dili in only 1 to 2 cases ( haloperidol , melperone , chlorprothixene , quetiapine , perazine , levomepromazine , promethazine , and risperidone ) . valproic acid was responsible for 3 dili cases followed by carbamazepine , galantamine , pregabaline , and lamotrigine , implicated in only one case each . maximum gamma - gt and transaminase ( glutamat - oxalat - transaminase [ got ] and glutamat - pyruvat - transaminase [ gpt ] ) values per dili case were evaluated for the time period from 2003 to 2011 ( values for agomelatine from 2009 to 2011 , as agomelatine was introduced in 2009 ) . as there are small deviations in terms of maximum got ( also known as aspartate - aminotransferase or ast ) , gpt ( also known as alanin - aminotranferase or alt ) , and alkaline phosphatase values across the participating institutions , a 5-fold increase in enzyme values was determined as dili . from 2003 on , measurement of liver enzymes was done at all participating hospitals at a temperature of 37c . prior to this , measurement was done at 15 to 20c , resulting in lower values for varying time periods at the different hospitals . duloxetine , clomipramine , and paroxetine were mainly responsible for high gpt values , while mirtazapine affected gpt values least . in terms of got values , duloxetine and clomipramine performed worst , and again mirtazapine had the least influence on got values . regarding -gt , duloxetine performed best , while trimipramine , clomipramine , as well as venlafaxine increased -gt values most ( figure 3a - c ) . the duration of treatment when dili occurred was different among the antidepressants ; mianserine was taken for 22 days on average , while mirtazapine was taken for 40 days . ( a ) gamma - glutamyl - transferase ( gamma - gt ) mean maximum values of single substances ( imputed alone for a minimum of 3 times except agomelatine due to its delayed implementation , which was imputed 2 times ) . ( b ) glutamat - oxalat - transaminase ( got ; also known as aspartate - aminotransferase [ ast ] ) mean maximum values of single substances ( imputed alone for a minimum of 3 cases , except agomelatine due to its delayed implementation , which was imputed 2 times ) . ( c ) glutamat - pyruvat - transaminase ( gpt ; also known as alanin - aminotransferase [ alt ] ) mean maximum values of single substances ( imputed alone for a minimum of 3 cases except agomelatine due to its delayed implementation ) . for inpatients with no preexisting liver damage , the mean maximum values for -gt , got , and gpt were 240 , 202 , and 285u / l , respectively , when dili was diagnosed . cases with preknown liver damage presented maximum -gt , got , and gpt mean values of 525 , 402 , and this indicates that preknown liver damage inpatients had more than doubled mean maximum values for -gt transaminases than subjects with normal liver status at the time when dili appeared . in our study sample , preexisting hepatic injury was the most common risk factor by far ( 59 cases ) , followed by substance abuse , mostly alcohol ( 20 cases ) . the most common clinical symptoms were nausea , fatigue , loss of appetite , and abdominal pain . a total of 27 inpatients showed clinical symptoms , while the majority did not show any . in 8 cases , the ad treatment remained and dosage was reduced , while in all other cases the drug was withdrawn after dili was assessed . within 55 cases , in our study sample of 149 liver enzyme elevations , only one case of acute liver failure occurred in a 20-year - old woman with a predamaged liver resulting from an overdose of paracetamol . at the time of admission to the psychiatric ward , she had been on a medication of 150 mg doxepine ( for 3 days ) and 10 mg olanzapine ( for 6 days ) . the patient s liver enzymes increased rapidly , and clinical symptoms such as vomiting , nausea , and epigastric pain set in . in the following laboratory analysis , a hepato - toxicity was identified ( bilirubin 3.8mg / dl , gpt 8827u / l , got 7363u / l , lactate dehydrogenase as soon as acute liver failure was diagnosed , the patient was transferred to the intensive care ward where she was under the care of the transplantation consulting team . the hepatotoxic effects of doxepine and olanzapine have been discribed in previous literature , but to our knowledge such a severe case has not been presented so far . to date , studies on the occurrence of the elevation of liver enzymes during psychotropic treatment have generally been based on case reports . a systematic drug surveillance program , however , increases the methodological accuracy significantly , and several such programs have shown links between adrs and a range of psychotropic drugs ( grohmann et al . , 2004 , 2013 ; gallego et al . , 2012 ; lettmaier et al . , 2012 ) . in our study , this result regarding the tcas is in accordance with the previous results of the amsp and arzneimittel - berwachungs - programm in der psychiatrie ( german drug surveillance in psychiatry ) study group . the amsp group published a manuscript on severe adrs of ads in the year 2004 ( degner et al . , 2004 ) . ads were classified according to receptors and their diverse action profiles , and tcas were linked to increased levels of liver enzymes . predominantly , these adrs provoke cholestatic liver damage with prolonged cholestasis , and hepatocellular necrosis may also occur ( zimmerman , 1999 ) . in an intensive drug monitoring study by the arzneimittel - berwachungs - programm working group , elevated liver values were observed in 13.8% of inpatients taking tcas , but the majority of inpatients presented with only minor increase in transaminases ( eg , gpt and ap in one - third of cases observed ) ( grohmann et al . , 1999 , 2004 ; degner et al . , 2004 ) . most tcas do not induce or inhibit cyp-450-isoenzymes . as a substrate of these enzymes , however , they may be affected by interactions , a point that is of interest due to their relatively restricted therapeutic index ( chou et al . up to 0.02% of inpatients receiving long - term therapy with fluoxetine showed elevated liver enzymes . while severe hepatotoxic reactions are rare , the literature reported some adrs linked to fluoxetine and a few to paroxetine and sertraline ( grohmann et al . , 1999 , 2004 ; many new ads inhibit cyp-450 enzymes ; for example , both fluoxetine and paroxetine are inhibitors of cyp2d6 . in combination with tcas , severe intoxications may occur , and in those involving 3 or more substances , the likelihood of toxicity is even higher ( gillman et al . , 2007 ) . as seen in short - term studies , mirtazapine elevates liver enzymes up to 3 times of the norm in 2% of patients , but in most cases patients do not develop significant liver damage , with some patients values even recovering in spite of continued medication ( hui et al . , 2002 ; biswas et al . , 2003 ) . two cases have been documented , however , in which mirtazapine induced severe cholestatic hepatitis ( dodd et al . , 2001 ; hui et al . , 2002 ) . within our study sample , mirtazapine did not perform worse than snris , especially in terms of gpt and got values , where it actually showed a favorable profile . in our study in cases of dili , the most prevalent drug class combination was the one of ads and aps , with most cases concerning a combination of ad with olanzapine or clozapine . a total of 5% to 10% of patients are slow metabolizers and show both high plasma levels and a high risk of a hepatotoxic reaction ( kevin et al . , 2007 ) . there is little information available on the newer generation of aps regarding hepatotoxic side effects , but extreme hepatotoxicity seems to occur very rarely . clozapine and risperidone induced liver damage , and even acute liver failure associated with clozapine has been documented ( macfarlane et al . , 1997 ) . olanzapine seems to trigger a hypersensitivity reaction with involvement of the liver ( mansur et al . clozapine causes a mild and mostly temporary increase in transaminases in 37% of patients ( grohmann et al . , 1989 ; our results are to some extent consistent with preexisting findings as summarized in a recent review of antidepressant - induced liver injury published in 2014 , which also indicated a greater risk of hepatotoxicity for tcas and agomelatine and the least potential for dili with ssris ( voican et el . , 2014 ) . the latter review claimed aminotransferase surveillance ( gpt ) as the most useful tool for detecting dili . ( 2014 ) , duloxetine and tcas such as clomipramine had the least favorable influence on gpt values . this is in agreement with our findings ; in our sample , the median dosage when dili occurred was higher than the overall median dosage in 7 of 9 substances . additionally , compared with existing findings , nefazodone and mao - inhibitors were often described as highly responsible for dili in previous studies , which can not be confirmed within the results of this surveillance program , as single mao inhibitors as well as nefazodone were only rarely prescribed and therefore could not be reliably compared with other drugs . our findings suggest that ssris are less likely than the other antidepressants examined in this study to precipitate dili . preknown liver damage inpatients are more at risk and had more than doubled mean values for -gt and transaminases than subjects with healthy liver status , at the time when dili appeared in our data . thus , special attention should be given to these inpatients when prescribing antidepressants with potential adverse effects affecting the liver . given the huge sample size in our observational naturalistic study , the present findings may contribute significantly to the existing literature and help to prevent antidepressant - induced adverse hepatic events . the findings from the present study reflect data obtained from inpatients who are likely to be more severely ill and have higher antidepressant dosages or more polypharmacy compared with outpatients . second , the detection of dili was dependent on increased liver enzyme values and hence on blood examination tests . regular blood tests are taken at the time of admittance to the hospital ; however , there is no standardized regimen for laboratory testing after admittance that might influence the detection of dili , especially in cases of asymptomatic drug - induced liver dysfunction . small differences in surveillance habits of liver enzymes across the 80 hospitals participating in the amsp program may further contribute to the aforementioned problem . this leads to a lower incidence rate of dili compared with other studies using gpt values 3 times and got values 2-fold above the normal value as indicative of dili . furthermore , reporting bias can not be ruled out due to the nature of the surveillance program . to prevent discrepancies among reported cases , the latter are discussed and examined in a systematic way at regional and international meetings within the amsp group . in terms of the results for agomelatine , it has to be mentioned that there was an awareness of possible liver adrs from the beginning of the surveillance . ( product safety information ) might have influenced the detection of agomelatine - induced liver enzyme elevations due to this sensitization prior to the onset of dili . the findings from the present study reflect data obtained from inpatients who are likely to be more severely ill and have higher antidepressant dosages or more polypharmacy compared with outpatients . second , the detection of dili was dependent on increased liver enzyme values and hence on blood examination tests . regular blood tests are taken at the time of admittance to the hospital ; however , there is no standardized regimen for laboratory testing after admittance that might influence the detection of dili , especially in cases of asymptomatic drug - induced liver dysfunction . small differences in surveillance habits of liver enzymes across the 80 hospitals participating in the amsp program may further contribute to the aforementioned problem . the amsp program focuses on only severe adrs ( grohmann et al . , 2004 ) with at least 5-fold increase of liver enzymes . this leads to a lower incidence rate of dili compared with other studies using gpt values 3 times and got values 2-fold above the normal value as indicative of dili . furthermore , reporting bias can not be ruled out due to the nature of the surveillance program . to prevent discrepancies among reported cases , the latter are discussed and examined in a systematic way at regional and international meetings within the amsp group . in terms of the results for agomelatine , it has to be mentioned that there was an awareness of possible liver adrs from the beginning of the surveillance ( product safety information ) might have influenced the detection of agomelatine - induced liver enzyme elevations due to this sensitization prior to the onset of dili . since 1993 educational and research grants have been given by the following pharmaceutical companies to the 3 local nonprofit associations of the amsp : ( 1 ) austrian companies : aesca pharma gmbh , astrazeneca sterreichgmbh , boehringer ingelheim austria , bristol myers squibb gmbh , csc pharmaceuticals gmbh , eli lilly gmbh , germania pharma gmbh , glaxosmithkline pharma gmbh , janssen - cilag pharma gmbh , lundbeck gmbh , novartis pharma gmbh , pfizer med inform , servier austria gmbh , and wyeth lederle pharma gmbh ; ( 2 ) german companies : abbott gmbh & co. kg , astrazeneca gmbh , aventis pharma deutschland gmbh ge - o / r / n , bayer vital gmbh & co. kg , boehringer mannheim gmbh , bristol - myers - squibb , ciba geigy gmbh , desitin arzneimittel gmbh , duphar pharma gmbh & co. kg , eisai gmbh , esparma gmbh arzneimittel , glaxosmithkline pharma gmbh & co. kg , hoffmann - la roche ag medical affairs , janssen - cilag gmbh , janssen research foundation , knoll deutschland gmbh , lilly deutschland gmbh niederlassung bad homburg , lundbeck gmbh & co. kg , novartis pharma gmbh , nordmark arzneimittel gmbh , organon gmbh , otsuka - pharma frankfurt , pfizer gmbh , pharmacia & upjohn gmbh , promonta lundbeck arzneimittel , rhone poulenc rohrer , sanofi - synthelabo gmbh , sanofi - aventis deutschland , schering ag , smithklinebeecham pharma gmbh , solvay arzneimittel gmbh , synthelabo arzneimittel gmbh , dr wilmar schwabe gmbh & co. , thiemann arzneimittel gmbh , troponwerke gmbh & co. kg , upjohn gmbh , wander pharma gmbh , and wyeth - pharma gmbh ; and ( 3 ) swiss companies : ahp ( schweiz ) ag , astrazeneca ag , bristol myers squibb ag , desitin pharma gmbh , eli lilly ( suisse ) s.a . , essex chemie ag , glaxosmithkline ag , janssen - cilag ag , lundbeck ( suisse ) ag , mepha schweiz ag / teva , msd merck sharp & dohme ag , organon ag , pfizer ag , pharmacia , sandoz pharmaceuticals ag , sanofi - aventis ( suisse ) s.a . , sanofi synthelabo sa , servier sa , smithklinebeecham ag , solvay pharma ag , vifor sa , wyeth ahp ( suisse ) ag , and wyeth pharmaceuticals ag . dr konstantinidis received honoraria from affiris , astrazeneca , novartis , pfizer , and servier , served as a consultant for astrazeneca , and was a speaker for astrazeneca , bristol myers squib , and janssen . dr winkler has received speaker honoraria from angelini , bristol - myers squibb , novartis , pfizer , and servier . dr greil has been a member of an advisory board for lundbeck and has received speaker s fees from astrazeneca , lundbeck , and lundbeck institute . myers squibb , eli lilly , glaxosmithkline , lundbeck , organon , sepracor , and servier ; he has served as a consultant or on advisory boards for astrazeneca , bristol myers squibb , eli lilly , glaxosmithkline , janssen , lundbeck , merck sharp and dome ( msd ) , novartis , organon , pfizer , schwabe , sepracor , and servier ; and has served on speakers bureaus for angelini , astrazeneca , bristol myers squibb , eli lilly , janssen , lundbeck , pfizer , pierre fabre , schwabe , sepracor , and servier . dr winkler has received lecture fees from bristol - myers squibb , csc pharmaceuticals , novartis , pfizer , and servier .

titanium and titanium alloys are currently accepted as the gold standard in dental applications . their excellent biocompatibility has been attributed to the inert titanium surface through the formation of a thin native oxide which has been correlated to the excellent corrosion resistance of this material in body fluids . whether this titanium oxide layer is essential to the outstanding biocompatibility of titanium surfaces in orthopedic biomaterial applications is still a moot point . to study this critical aspect further , human fetal osteoblasts were cultured on thermally oxidized and microarc oxidized ( mao ) surfaces and cell differentiation , a key indicator in bone tissue growth , was quantified by measuring the expression of alkaline phosphatase ( alp ) using a commercial assay kit . cell attachment was similar on all the oxidized surfaces although alp expression was highest on the oxidized titanium alloy surfaces . untreated titanium alloy surfaces showed a distinctly lower degree of alp activity . this indicates that titanium oxide clearly upregulates alp expression in human fetal osteoblasts and may be a key bioactive factor that causes the excellent biocompatibility of titanium alloys . this result may make it imperative to incorporate titanium oxide in all hard tissue applications involving titanium and other alloys .

titanium alloys have become the most popular metallic biomaterials in dental applications because of their excellent biocompatibility . this is attributed to the inert nature of the titanium surface due to the formation of a thin native titanium oxide layer which also provides excellent corrosion resistance . although titanium alloys have virtually replaced other metallic biomaterials in dental implant applications , currently there is little insight into the reasons for this excellent biocompatibility of titanium surfaces . a number of studies have pointed out various factors that contribute to the biocompatibility of titanium or of modified titanium surfaces [ 36 ] . while these studies have investigated both proliferation and differentiation of osteoblast cell lines evidenced in many instances by gene expression to corroborate biocompatibility , the emphasis has been on physical factors such as roughness , texture , wettability , or substrate microstructural features . some importance has been paid to the substrate composition , whether ti or ti alloy , and the makeup of the surface modified layer . in recent work , the plausible role of titanium oxide in contributing to this outstanding biocompatibility was observed . in studying titanium alloys , it was noticed that alp activity was higher on oxidized titanium alloys compared to corresponding untreated materials [ 7 , 8 ] . despite having a good understanding of the signaling pathways in osteoblast differentiation , the effect of titanium oxide on osteoblast differentiation has not been fully researched even though the fact that a thin native titanium oxide layer forms on all titanium alloys is well known and a large amount of research has been conducted on these popular biomaterials . it is still unclear at the present time whether osteoblast differentiation is affected by titanium oxide or by the oxygen released from the titanium oxide . a recent report suggests that oxygen tension , in and of itself , has a strong effect on osteoblast differentiation and may in fact regulate this process , and hypoxic cell cultures demonstrated a lower level of mineralization resulting in a more chondrogenic tissue in comparison to higher levels of oxygen in cell cultures . healing of fractures has also been reported much earlier to be delayed in the absence of oxygen . in contrast , reactive oxygen species ( ros ) has been reported to suppress bone formation and stimulate bone resorption . osteoblast differentiation involves a complex molecular pathway consisting of various transcription factors and it is well known that many transitional stages comprise the pathway for this process and that several signaling molecules play key roles in overall skeletal development . it is currently unknown if titanium oxide plays a critical role in any step of the signaling cascade . understanding the manner in which titanium oxide affects osteoblast differentiation may be critical in titanium implantology in terms of reducing hospitalization time and formulating efficient therapeutic procedures . in this study , hfob cells were cultured for 10 days on the oxide formed on the surface of two titanium - based alloys from two different methods of oxidation and the degree of osteoblast differentiation was measured through quantification of alp activity using a commercial assay kit to compare with unoxidized titanium alloy surfaces in an effort to determine if titanium oxide indeed played a role in the differentiation process . various samples of the two titanium - based alloys , gamma - tial ( -tial [ ti-48al-2cr-2nb ( at.% ) ] ) and ti-6al-4v ( wt.% ) , were machined in the form of 7 mm diameter cylindrical rods . from these , disks with an approximate thickness of 1 mm were obtained using a slow - speed diamond saw ( buehler ) . both surfaces of the disks were ground using 240 , 320 , 600 , and 1200 grit silicon carbide paper in an ecomet 3 ( buehler ) . these metal disks were then sonicated in 0.8% alconox ( fisher , pittsburgh , pennsylvania ) and 70% ethanol for 10 minutes each , rinsed with deionized water , and dried with a hot - air blow - dryer . both -tial and ti-6al-4v disks were oxidized in a laboratory furnace ( cm furnaces inc . ) in air at 500c or 800c for 1 h and later placed in 48-well cell culture plates ( corning ) . the nomenclature followed in this paper is as follows : -tial and ti-6al-4v disks oxidized at 500c and 800c are hereafter referred to as gti5 , gti8 , tiv5 , and tiv8 , respectively , while untreated disks are designated as gti and tiv . atomic force microscopy ( afm ) was used to obtain average surface roughness values of the oxidized surfaces . other -tial and ti-6al-4v disk samples were processed using micro arc oxidation ( mao ) . for the mao process , a stainless steel beaker was used as the cathode , while the titanium disk ( either -tial or ti-6al-4v ) was used as the anode . each sample was mounted in a titanium holder specially designed to allow complete exposure of the sample to the electrolyte . a hoefer ps300-b high voltage power supply ( 300 v ; 500 ma ) was operated in galvanostatic mode in order to form the titanium oxide film on the sample surface using the mao process . process conditions of sample current of 200 ma and 225 ma for durations of 3 and 4 minutes for each case were utilized based on an earlier study . after treatment , the samples were rinsed with distilled water and then dried with a blow dryer . the micro arc oxidized samples will be henceforth referred to as maogti for the -tial samples and the maotiv for the ti-6al-4v samples , respectively . the oxides formed on both -tial and ti-6al-4v as a result of the mao treatment for the applied process conditions are mainly rutile and anatase as reported earlier [ 11 , 12 ] . for thermal oxidation , alumina is the dominant oxide formed on -tial at 500c , while rutile is dominant at 800c [ 1316 ] . for ti-6al-4v , thermal oxidation at 500c produces a combination of an oxide diffused ti(o ) and rutile where the latter phase appears to grow at high temperatures between 650c and 800c . the average roughness values of these surfaces were extracted from topography analysis using afm and presented in table 1 . human osteoblast cells , cell line hfob 1.19 ( atcc , manassas , virginia ) , were cultured in 90% dulbecco 's modified eagle 's medium nutrient mixture f-12 ham ( dmem ) ( sigma - aldrich , st . louis , missouri ) with 2.5 mm l - glutamine and 15 mm hepes , without phenol red , supplemented with 0.3 mg / ml g-418 ( calbiochem , san diego , california ) and 10% fetal bovine serum ( fbs ) ( hyclone , logan , utah ) . cells were grown in 25 cm plastic culture flasks ( corning , corning , new york ) and incubated at 33.5c until confluence . at approximately 100% confluence , cells were washed three times with phosphate buffer saline ( pbs ) solution ( 137 mm nacl , 2.7 mm kcl , 4.3 mm na2hpo4 , and 1.4 mm kh2hpo4 ) and harvested using 0.25% trypsin-0.53 mm edta ( gibco , gaithersburg , maryland ) at 37c for 5 min . cells were then pelleted by low speed centrifugation ( 3,300 rpm ) for 5 minutes and subcultured at a 1 : 3 ratio . cells were seeded in 48-well plates ( becton , dickinson , lincoln park , nj ) at a density of 5 10 cells / cm on tiv , tiv5 , tiv8 , gti , gti5 , gti8 , maotiv , and maogti disks ( 7 mm in diameter ) , using the commercial alkaline phosphatase colorimetric assay kit ( ab83369 , abcam ) in order to evaluate osteoblast differentiation quantitatively on thermally oxidized , micro arc oxidized , and untreated -tial and ti-6al-4v disks . samples were incubated for 3 days at 33.5c and then for 7 days at 39.5c to allow osteoblast differentiation . modifications to the suggested protocol were made to achieve a more efficient cell lysis , including washing the samples carefully three times with pbs and homogenizing in 60 l of the assay buffer . also , triton x-100 ( 80 l ) was utilized to lyse the cells for an efficient measurement of intracellular alp . stop solution ( 20 l ) was added to terminate alp activity in the sample . the solution in each well was transferred to a 96-well plate ( becton , dickinson , lincoln park , nj ) . pnpp solution ( 50 l ) was added to each well containing the test samples and background controls . a standard curve was generated to determine the concentration of alp activity in the sample for which 40 l of the 5 mm pnpp solution was diluted with 160 l assay buffer to generate a 1 mm pnpp standard . 0 , 4 , 8 , 12 , 16 , and 20 l were added to 96-well plate in duplicate to generate 0 , 4 , 8 , 12 , 16 , and 20 nmol / well pnpp standard . alp enzyme solution ( 10 l ) was added to each well containing the pnpp standard . all reactions were stopped by adding 20 l stop solution to each standard and sample reaction except the sample background control reaction ( since 20 l stop solution had been added to the background control when prepared previously ) . the background was corrected by subtracting the value derived from the zero ( 0 ) standards from all standards , samples , and sample background control . the pnpp standard curve was plotted and the sample readings were applied to the standard curve to get the amount of pnpp generated . alp activity of the test samples was calculated using the equation , alp activity ( u / ml ) = a / v / t , where a is amount of pnpp generated by samples ( in mol ) , v is volume of sample added to the assay well ( in ml ) , and t is reaction time ( in minutes ) . three independent experiments were performed for each alp assay , and since each experiment had three replicates , a total of nine replicates per surface were evaluated ( mao -tial , thermally oxidized -tial , untreated -tial , mao ti-6al-4v , thermally oxidized ti-6al-4v , untreated ti-6al-4v , and control glass coverslips ) for a 10-day period of culture . the data from the alp assay is presented as the mean standard deviation ( sd ) of the optical density of differentiated cells on the different surfaces corresponding to the amount of alkaline phosphatase detected . each value represents the mean of three measurements of cell differentiation performed on a specific surface as mentioned above . a factorial analysis of variance ( anova ) was used to assess the significant interactions between type of metal ( -tial or ti-6al-4v ) and type of surface treatment ( micro arc oxidization at 200 ma , 3 min , 200 ma , 4 min , 225 ma , 3 min , and 225 ma , 4 min ; thermal oxidization at 500c and 800c ) . all significant interactions were graphically analyzed and , in addition , a randomized block design was performed to reduce the variance in the data . furthermore , a contrast test was performed to compare the type of metal ( -tial and ti-6al-4v ) with the surface treatments . significant differences in cell differentiation on the type of metal and surfaces tested were confirmed using the lsd fisher test . sem images of glass coverslips , untreated ti-6al-4v and -tial surfaces ( tiv and gti ) , micro arc oxidized ti-6al-4v and -tial surfaces ( maotiv and maogti ) , and thermally oxidized ti-6al-4v and -tial surfaces ( tiv5 , tiv8 , gti5 , and gti8 ) , are shown in figure 1 . gti and tiv ( figures 1(a ) and 1(b ) ) exhibit a smoother surface of the passive oxide layer formed instantaneously on ti alloys . in contrast , rounded surface structures were visible in tiv5 , suggesting clusters of oxide granules . gti8 and tiv8 , on the other hand , exhibited a rougher surface in comparison to the other samples ( figures 1(e ) and 1(f ) ) . larger oxide granules were present on tiv8 ( figure 1(e ) ) compared to those on gti8 ( figure 1(f ) ) , conferring an irregular appearance to this surface and suggesting that tiv8 oxide layer could possibly be thicker . the mao surfaces for ti-6al-4v ( maotiv ) ( figure 1(g ) ) demonstrated a number of large pores on the oxide surface typical of similar treatments on ti alloys [ 18 , 19 ] . although pores were also clearly visible on the surface of maogti , these were smaller and on the average in the submicron range . sem images shown in figure 2 indicate that hfob 1.19 cells grew normally on the surfaces of untreated -tial and ti-6al-4v disks . cell attachment and proliferation were similar on both metal surfaces , suggesting normal growth , cell confluence , and attachment under in vitro conditions . the osteoblast cells were spread and flattened on the glass coverslips exhibiting such close contact with each other , that detection of cellular boundaries was difficult . fibrous networks corresponding to fibrillar collagen , the main component of bone ecm , which aid in the adhesion of cells and important for the proper assembly of the extracellular matrix , are visible lending further testimony to the normal growth of osteoblasts on these surfaces . the ecm , which serves as a calcium phosphate reservoir , provides support for the cells , offers protection , and is very important in homeostasis , appears to be forming copiously . included are nodules of mineralization with a sponge - like morphology and intimately associated with the fibrillar network and scattered throughout the samples . the maturation of the ecm is evidenced by the presence of fibrous networks associated with cells and an increase in nodules of mineralization . on the thermally oxidized ti-6al-4v and -tial alloys at 500c , cellular attachment and proliferation were similarly observed . a cell multilayer , constituted by elongated and polygonal cells with some round shaped cells ( figure 2 ) , along with the presence of a few small rounded structures which may correspond to mineral nodules , was observed . at higher magnification ( figure 3 ) , the mineral nodules appeared to be in close contact with cells and had a sponge - like appearance . on the thermally there was cell debris indicative of cytotoxicity of the oxide ( figure 3 ) in agreement with an earlier report . however , on thermally oxidized -tial alloys , fibrous networks and mineralized nodules were observed on elongated cells ( see figure 3 ) . slender cytoplasmic projections ( filopodia ) extended from the cells in all directions on all micro arc oxidized ti-6al-4v and -tial disks , confirming the biocompatibility of the substrate materials . in addition , sheet - like cytoplasmic protrusions extending from the cell body in all directions suggest the ability of cellular movement , spreading of the cells on the substrate , and/or the fact that cellular division ( mitosis ) may be occurring . the presence of mineralized nodules again suggests the maturation of the ecm and the formation of bone - like tissue indicating osteoblast differentiation evidenced by a high degree of alp activity . taken together , normal cell attachment and proliferation on all the surfaces with the exception of tiv8 indicate the excellent biocompatibility of control , untreated , and treated titanium alloy surfaces . standard calibration curves were used to extrapolate the values of alkaline phosphatase ( alp ) activity on experimental disks 10 days after seeding based on the alkaline phosphatase assay . the alp activity , measured as described in section 2 , is plotted in figure 4 for the various sample surfaces that were utilized in the experiment to measure osteoblast differentiation . it is clear that little alp activity is observed on the untreated ti alloy samples while the positive controls ( glass coverslips ) indicate reasonable alp activity corresponding to osteoblast differentiation . for the surface treatments of thermal oxidation and mao , the highest alp activity is observed for the ti alloys subjected to the mao treatment . as an exception , it must be noted that the alp activity on ti-6al-4v oxidized at 800c is rather low compared to the other treated samples . anova revealed significant interactions between the factors tested ( type of metal and treatment ) and alp activity . the interaction between the type of metal ( -tial or ti-6al-4v ) and surface treatment ( micro arc oxidation at 200 ma , 3 min , 200 ma , 4 min , 225 ma , 3 min , and 225 ma , 4 min ; thermal oxidation at 500c or 800c ) was significant ( p < 0.05 ) . there were also significant differences in the amount of alkaline phosphatase detected among these six surfaces studied after 10 days of incubation at 33.5c and 39.5c , respectively . additionally , alkaline phosphatase activity was lower on the positive control ( glass coverslips ) and even much lower on untreated titanium alloy surfaces in comparison with the micro arc and thermally oxidized alloys . furthermore , alp activity increased in -tial and ti-6al-4v alloys and in the mao treatments where the samples were exposed for longer process times to current density . although qualitatively there were no significant differences in the number of osteoblast cells attached on the micro arc oxidized or thermally oxidized surfaces collectively , alp activity was significantly higher on the cell cultures that grew on the micro arc oxidized surfaces in comparison to the thermally oxidized substrates ( see figure 4 ) . surface roughness of the oxidized surfaces does not appear to show a correlation with alp activity . hfob adherence is clearly excellent on all surfaces with the exception of tiv8 which may possibly be due to the cellular response to toxic compounds or harmful ions such as vanadium in the oxide layer as a result of thermal oxidation . as such , titanium oxide alone does not pose problems of cytotoxicity since cells did attach and proliferate on surfaces of both alloys subject to oxidation at 500c and also -tial oxidized at 800c where the oxide formed is predominantly composed of titanium oxide in the form of rutile or anatase . an earlier study showed normal cell attachment on tiv8 2 days after seeding but cell debris as a result of subsequent cell death for longer periods of incubation . osteoblast differentiation , on the other hand , occurred to different extents on all the substrates as measured by alp activity . osteoblast differentiation is a well - coordinated physiological process occurring in three stages which include cell proliferation , ecm production and maturation , and matrix mineralization . correspondingly , proliferative osteoprogenitors such as msx2 and rp59 are expressed in the first stage , followed by runx2 , osx , and oc ( osteocalcin ) during the later stages in this process of differentiation . during proliferation , col 1 and alp are detected earlier on followed by the secretion of rgd containing proteins such as bone sialoproteins ( bsp ) and osteopontin ( op ) and culminating in the synthesis of oc in the last stage of differentiation . bone morphogenetic proteins ( bmps ) and various members of tgf- family are secreted by the osteoblast cells and , once sequestered in the extracellular matrix ( ecm ) , these have also been reported to be critical for osteoblast differentiation . interaction between type i collagen and 21 integrin activates a mitogen - activated protein kinase ( mapk ) pathway which results in the phosphorylation and activation of cbfa1 , in turn stimulating the differentiation of osteoblasts . , the lack of expression of runx2 and osx may result in the formation of demineralized bone , although data suggests that these transcriptional factors act independently of each other . expression of runx2 along with cbf and alkaline phosphatase has been found to be critical in the early stage of differentiation while osx becomes very important in the later stage of osteoblast differentiation . while , in the present study , cell attachment and proliferation occur normally on all the substrates , except for tiv8 , alkaline phosphatase activity measurements indicate that osteoblast differentiation varies depending on the nature of the substrate . the presence of mineral nodules in the ecm on the titanium sample surfaces provides physical evidence to corroborate the activity of osteoblasts in one of their functional stages after maturation as observed in osteoblast cultures at longer periods of incubation . ti-6al-4v and -tial thermally oxidized disks at 500c showed both irregular and rounded mineralized structures on the surface . similar cell morphology and function were observed for the mao surfaces for both alloys , suggesting the ability of these surfaces to also promulgate differentiation . the fact that alp activity was significantly lower on the untreated alloys ( tiv , gti ) suggests that the titanium oxide formed on the surface of these alloys may be strongly correlated with the differentiation of the osteoblasts . it was demonstrated in an earlier study that the oxide layer on mao treated alloys consists of titanium oxide with peaks for both anatase and rutile phases in all the coating conditions applied in this study . both anatase and rutile have been shown to be beneficial in enhancing nucleation and subsequent hydroxyapatite ( ha ) precipitation , thereby increasing bioactivity of the titanium surface . the results from the present study also suggest that the incorporation of calcium ( ca ) and phosphorus ( p ) into the titanium oxide may be favorable for cell differentiation . while ca and p are important in the observation of increased alp activity on mao treated surfaces , the ti alloys samples which were thermally oxidized at 500c ( devoid of ca and p ) also show a reasonably high alp activity compared to control glass coverslips . a recent study has shown higher elemental oxygen concentration and higher water wettability on tio2 surfaces when compared to bare titanium surfaces resulting in a twofold increase in alp activity and mineralized nodule area . it also appears that the topography of bioengineered titanium surfaces affects gene expression and phenotypic response of osteoprogenitor cells . higher alp activity on surfaces containing titanium oxide may possibly be correlated with the surface topography of the substrate which may affect cell proliferation and differentiation [ 31 , 32 ] . thus it may be argued that the osteoblast differentiation does not depend solely on ca and p ions but more so on the presence of titanium oxide . in contrast , another research suggests cell cytotoxicity due to tio2 resulting from the interaction between tio2 nanoparticles and the lysosomal compartment , independently of the known apoptotic signaling pathways . in addition , tio2 has been reported to possess antibacterial characteristics in stark contrast to its positive biocompatibility . although it is clear from this study that titanium oxide increases the alp activity in osteoblasts , the mechanism associated with this process is still unknown . it was proposed that bmp-2 controls alkaline phosphatase expression and osteoblast mineralization by a wnt autocrine loop in mesenchymal stem cells ( mscs ) . among the factors that regulate msc growth and differentiation are soluble factors and cell - substrate interactions , although little is known about the molecular mechanisms by which soluble and substrate signals regulate msc function . these authors showed that the commitment of human mscs to the osteogenic and adipogenic lineages in vitro involves signaling by mitogen - activated protein kinase ( mapk ) pathways . in particular , it was found that dexamethasone , ascorbic acid , and -glycerophosphate induce msc differentiation by regulating the extracellular signal - regulated kinase ( erk1/2 ) cascade . furthermore , blocking the erk1/2 pathway inhibits osteogenic differentiation of mscs and leads to adipogenesis . thus mapk pathways , which are generally activated by growth factors / cytokines and integrin - mediated cell adhesion , play a critical role in directing msc commitment . mapk pathways are also activated by physical stimuli to regulate the function of a variety of cell types , including bone cells . in bone , it has been proposed that mature cells such as osteocytes and osteoblasts are responsible for sensing and responding to mechanical stimuli . it is unknown whether the progenitors that give rise to these cells are responsive to mechanical signals . various signaling pathways , including bmp , wnt , and notch , regulate bone homeostasis . it is difficult at the present time to determine which of these is affected by titanium oxide to the extent of upregulating alp activity . while it is clearly demonstrated that hypoxia suppresses osteoblast differentiation and as a consequence bone formation , the mode by which tio2 increases alp activity is not clear cut . one may speculate that a chemical reaction between tio2 and the culture medium may somehow result in the release of oxygen and this normoxia has a positive impact on osteoblast differentiation . however , it is clear that tio2 is a bioactive factor which indeed upregulates alp activity , although the manner in which the oxide indeed participates in the biochemical signaling cascade which occurs during differentiation does require further study . this information may be vital in the future of implantology in accelerating fracture healing and other tissue regenerative processes in dental and orthopedic applications . if titanium oxide indeed upregulates osteoblast differentiation , the manufacturers of titanium - based implants would find it advantageous to incorporate titanium oxide as a coating on every surface layer in contact with the tissue side of the implant . clearly , further research is needed to interrogate the empirical ability of titanium oxide to preferentially favor osteoblast differentiation or at least alp activity . alp activity is much higher on oxidized surfaces of both titanium alloys compared to untreated surfaces most probably due to the presence of titanium oxide.the higher alp activity on micro arc oxidized surfaces is attributed to the ca and p content which are not present in the thermally oxidized titanium alloys.the mechanism for the upregulation of alp due to titanium oxide needs further study although it is clear that tio2 is a bioactive factor in osteoblast differentiation . alp activity is much higher on oxidized surfaces of both titanium alloys compared to untreated surfaces most probably due to the presence of titanium oxide . the higher alp activity on micro arc oxidized surfaces is attributed to the ca and p content which are not present in the thermally oxidized titanium alloys . the mechanism for the upregulation of alp due to titanium oxide needs further study although it is clear that tio2 is a bioactive factor in osteoblast differentiation .

background : initial research indicated that higher concentration of osteoprotegerin ( opg ) is associated with healthy periodontium ( protective ) and its concentration decreases as the periodontal disease progresses . however , till date , there are no studies to investigate the levels of opg in gingival crevicular fluid ( gcf ) after the treatment of periodontitis . hence , the present study was carried out to assess its concentration in gcf to find out their association if any , and to explore its possible use as a novel bone marker of the host modulation of periodontal disease.materials and methods : sixty - four subjects were divided into 4 groups ( 16 each ) , based on clinical attachment loss ( cal ) and radiological parameters ( bone loss ) ; healthy ( group i ) , gingivitis ( group ii ) , slight periodontitis ( group iii ) , and moderate - to - severe periodontitis ( group iv ) . moderate - to - severe periodontitis subjects , after nonsurgical periodontal treatment , ( srp ) constituted group v. gcf samples were collected to estimate the levels of opg using enzyme - linked immunosorbent assay ( elisa ) . the kruskal - wallis , man - whitney u test , and wilcoxon signed - rank tests were carried out to compare opg levels among groups . the spearman rank correlation test was used to correlate opg levels between the study groups and the clinical parameters ; p < 0.05 was considered significant.results:the highest mean opg concentration in gcf was obtained for group i ( 162.47 51.171 pg/ l ) and the least for group iv ( 10.92 1.913 pg/l ) , suggesting a negative correlation between opg concentration and cal . opg concentrations in gcf after the treatment of group iv increased from 10.92 1.913 pg/l to 15.63 4.679 pg/l.conclusion : opg concentration in gcf was inversely proportional to cal and not an active progression factor for periodontal disease . further , after the treatment of moderate - to - severe periodontitis subjects ( group iv ) , opg concentrations increased . hence , it can be concluded that opg could be considered as a novel bone marker the host modulation of periodontal disease .

periodontitis , a chronic inflammatory disease , is characterized by increased expression of various cytokines and other inflammatory mediators resulting in extensive osteoclast formation and bone loss . these cytokines affect bone remodeling and play a vital role in both the physiological and pathological regulation of bone . the level of bone mass which is essential to execute various functions is maintained by a balanced act of bone formation and bone resorption . this process is highly co - ordinated and regulated by two specialized cells , osteoblasts , the bone - forming cells and osteoclasts , the bone - resorbing cells . these cells are controlled by various hormones , inflammatory mediators , cytokines , and growth factors . recent addition to these regulators is the multifactorial cytokine opg which has been detected in gcf . opg , a key physiological inhibitor of osteoclastic bone resorption , is a glycoprotein which belongs to the tumor necrosis factor receptor ( tnf ) super family . it is a decoy receptor for receptor activator nuclear factor kappa b ligand ( rankl ) and inhibits cell - to - cell signaling between marrow stromal cells and precursors of osteoclast . thus , it prevents the bone resorption by rankl , which is a precursor for the production of osteoclasts . opg is expressed by various cell types like osteoblasts , osteoclastic stromal cells , t cells , b cells , chondrocytes , and follicular dendritic cells . however , it is also found in organs like the kidney , liver , heart , lung , spleen , thyroid , lymph nodes , thymus , brain , and placenta . it is also found in periodontal and dental tissues like gingiva and periodontal ligament , both in the internal and the external enamel epithelium , as well as in the mesenchyme of the dental papilla during tooth development . moreover , a prominent expression of opg in the cartilaginous primordia of developing maxilla , mandible , and hyoid bone has been reported . further , opg has been reported to have a preventive role in rheumatoid arthritis - associated bone erosion in joints and its deficiency in inflamed joints of rheumatoid arthritis confirms this finding . recently , opg was isolated in gingival tissue and gcf with its levels decreasing with the progression of periodontal diseases suggesting that opg levels in gcf may become a modulator of periodontal disease , especially alveolar bone resorption . however , till date , there have been no studies on the correlation of opg levels in gcf during periodontal health , disease , and after nonsurgical periodontal therapy ( srp ) . hence in the light of the aforementioned facts , this clinico - biochemical study was designed to estimate the levels of opg in the gcf of subjects with clinically healthy periodontium , gingivitis , slight periodontitis , moderate - to - severe periodontitis , and of moderate - to - severe periodontitis subjects after scaling and root planing ( srp ) . this study conducted during the period from august to september 2007 involved 64 subjects ( 32 females , 32 males ) aged 30 - 39 years selected from the outpatient section , department of periodontics , government dental college and research institute , bengaluru , karnataka , india . essential ethical clearance for the study was obtained from the institutional ethical review board , government dental college and research institute , rajiv gandhi university of health sciences , karnataka , india . those who volunteered were briefed about the study procedure , and a consent form was signed on acceptance by them . specific conditions that excluded participation in the study were ( i ) pregnant , lactating , and postmenopausal women , ( ii ) patients with diabetes mellitus , ischemic heart disease , or any other conditions contributing to atherosclerosis , malignant tumors , rheumatoid arthritis , bone disorders , and/or on cancer chemotherapy or those on antiresorptive drugs like bisphosphonates , with this disese will hve opg concentration less because of inflammation , ( iii ) smokers and alcoholics , ( iv ) subjects who received treatment with anti - inflammatory drugs , antibiotics , steroids , or contraceptives in the last six months , and ( v ) those receiving any periodontal treatment . the subjects were categorized into groups , each group comprising 16 patients based on modified gingival index ( mgi ) and clinical attachment loss ( cal ) with radiographic evidence of bone loss . group i ( healthy ) : consisted of 16 subjects with clinically healthy periodontium and with no evidence of disease . the score obtained after assessing the gingival status using mgi was zero and with no crestal bone loss as determined from the radiograph . group ii ( gingivitis ) : consisted of 16 subjects whose gingivae showed clinical signs of inflammation but there was no evidence of cal , which was zero . group iii ( slight periodontitis ) : consisted of 16 subjects , who showed clinical signs of gingival inflammation and cal of 1 - 2 mm with radiographic evidence of bone loss . group iv ( moderate to severe periodontitis ) : consisted of 16 subjects , who showed clinical signs of gingival inflammation and cal > 3 mm with radiographic evidence of bone loss . group v ( after - treatment group ) : consisted of group iv subjects treated nonsurgically , and gcf samples were collected from the same sites six to eight weeks after treatment , to constitute group v [ figures 15 ] . clinical picture of a patient of group i ( healthy ) showing cal of 0 mm on the mesiolabial aspect of 11 radiographic picture of same area clinical picture of a patient of group ii ( gingivitis ) showing cal of 0 mm on the mesiolabial aspect of 11 radiographic picture of same area clinical picture of a patient of group iii ( slight periodontitis ) showing cal of 1 - 2 mm on the mesiolabial aspect of 21 radiographic picture of same area clinical picture of a patient of group iv ( moderate to severe periodontitis ) showing cal of > 3 mm on the mesiobuccal aspect of 46 radiographic picture of same area clinical picture of a patient of group v ( moderate to severe periodontitis after treatment ) radiographic picture of same area after treatment the sampling protocol was followed as described previously in the earlier studies by the group . briefly , clinical and radiological examinations , group allocation , and sampling - site selections were performed by one examiner ( pb ) and the samples were collected on the subsequent day by the second examiner ( mvrp ) . this was done to ensure blinding of the sampling examiner and to prevent contamination of gcf with blood associated with the probing of inflamed sites . only one site per subject was selected as a sampling site . in the healthy group , sampling was predetermined to be from the mesiobuccal region of the maxillary right or left first molar , wherever adequate sample was obtainable . in gingivitis , , sites with 1 - 6 mm cal were identified using a williams graduated periodontal probe followed by radiographical confirmation of bone loss and assigned for sampling . on subsequent day , a standardized volume of 1 ml gcf was collected from each predetermined test site using the calibration on color - coded 1 - 5 ml calibrated volumetric microcapillary pipettes with an extracrevicular approach ( unstimulated ) . plastic vials were used to store the gcf - containing micropipettes and stored at 70c until the assay [ figures 6 and 7 ] . the microcapillary pipettes with aspirator tube collection of gcf using extra - crevicular positioning of microcapillary pipette after appropriate dilution of gcf samples , a sandwich type of elisa development kit ( elisa : enzyme - linked immunosorbent assay ) was employed for the quantitative determination of human opg [ figures 8 and 9 ] . duoset human opg elisa kit contents of opg elisa kit the 96 well - plate kit was prepared the previous day and incubated overnight , as per the manufacturer 's kit development instructions . the kit utilizes a capture antibody which was coated on a microtiter plate for immobilization and to bind the human opg in the standards or sample . then , 100 l of diluted sample and standards were added to appropriate wells and covered with adhesive strips followed by incubation for two hours at room temperature . later , 100 l ( each ) of working dilution of streptavidin - horseradish peroxidase ( hrp ) and substrate solution were added to each well , after washing the plate three times with wash buffer between each addition , and incubated for 20 minutes at room temperature . finally , 50 l of stop solution was added to each well to stop enzyme reaction and the color generated was read at 450 nm . the concentration of opg in the tested samples was calculated using the standard curve plotted using the optical density values with the standards [ figure 10 ] . opg capture antibody preparation a statistical software program was applied to analyze the data obtained . the kruskal - wallis , man - whitney u test , and wilcoxon signed - rank tests were carried out to compare opg levels among groups . the spearman rank correlation test was used to correlate opg levels between the study groups and the clinical parameters . the sampling protocol was followed as described previously in the earlier studies by the group . briefly , clinical and radiological examinations , group allocation , and sampling - site selections were performed by one examiner ( pb ) and the samples were collected on the subsequent day by the second examiner ( mvrp ) . this was done to ensure blinding of the sampling examiner and to prevent contamination of gcf with blood associated with the probing of inflamed sites . only one site per subject was selected as a sampling site . in the healthy group , sampling was predetermined to be from the mesiobuccal region of the maxillary right or left first molar , wherever adequate sample was obtainable . in gingivitis , sites with the highest clinical signs of inflammation were selected . in periodontitis , sites with 1 - 6 mm cal were identified using a williams graduated periodontal probe followed by radiographical confirmation of bone loss and assigned for sampling . on subsequent day , a standardized volume of 1 ml gcf was collected from each predetermined test site using the calibration on color - coded 1 - 5 ml calibrated volumetric microcapillary pipettes with an extracrevicular approach ( unstimulated ) . plastic vials were used to store the gcf - containing micropipettes and stored at 70c until the assay [ figures 6 and 7 ] . the microcapillary pipettes with aspirator tube collection of gcf using extra - crevicular positioning of microcapillary pipette after appropriate dilution of gcf samples , a sandwich type of elisa development kit ( elisa : enzyme - linked immunosorbent assay ) was employed for the quantitative determination of human opg [ figures 8 and 9 ] . duoset human opg elisa kit contents of opg elisa kit the 96 well - plate kit was prepared the previous day and incubated overnight , as per the manufacturer 's kit development instructions . the kit utilizes a capture antibody which was coated on a microtiter plate for immobilization and to bind the human opg in the standards or sample . then , 100 l of diluted sample and standards were added to appropriate wells and covered with adhesive strips followed by incubation for two hours at room temperature . later , 100 l ( each ) of working dilution of streptavidin - horseradish peroxidase ( hrp ) and substrate solution were added to each well , after washing the plate three times with wash buffer between each addition , and incubated for 20 minutes at room temperature . finally , 50 l of stop solution was added to each well to stop enzyme reaction and the color generated was read at 450 nm . the concentration of opg in the tested samples was calculated using the standard curve plotted using the optical density values with the standards [ figure 10 ] . the kruskal - wallis , man - whitney u test , and wilcoxon signed - rank tests were carried out to compare opg levels among groups . the spearman rank correlation test was used to correlate opg levels between the study groups and the clinical parameters . the highest mean opg concentration was noted in group i ( 162.47 51.17 pg/l ) and lowest in group iv , that is , 10.92 1.91 pg/l with intermediate values for group ii ( 41.39 16.64 pg/ l ) , group iii ( 23.40 1.99 pg/ l ) , and group v ( 15.63 4.68 pg/ l ) . mean opg concentration of the study groups graph comparing opg levels among study groups the results of kruskal wallis test ( nonparametric ) , carried out to evaluate the difference in opg levels with 5% level of significance ( p < 0.05 ) , suggested that the mean concentration of opg differed significantly among the groups tested [ table 2 ] . kruskal - wallis test comparing mean gcf opg levels between groups further , multiple comparison using mann - whitney u test carried out to find the pair or pairs of groups that differed significantly ( p < 0.05 ) suggested that opg levels in gcf decreased progressively from health to severe periodontitis [ table 3 ] . pairwise comparison using mann - whitney u test for gcf opg when group iv ( moderate to severe periodontitis ) and group v ( after treatment of group iv ) were compared using wilcoxon signed - rank test [ table 4 ] , the difference in the concentration of opg was statistically significant ( p < 0.05 ) , indicating that after srp , opg levels increased considerably in gcf ( 10.92 pg/l to 15.63 pg/l ) . wilcoxon signed - rank test comparing gcf opg concentration before and after treatment spearman 's rank correlation test was done to observe any correlation between the gcf opg concentration and clinical variables ( mgi and cal ) . when opg levels in gcf were tested for correlation with the disease severity measures , that is , mgi ( in group i and ii ) and mgi and cal ( in groups iii , iv , and v ) , a negative correlation was found for gcf opg concentration as shown in table 5 . kruskal -wallis test comparing cal and opg concentrations when kruskal wallis test was done to compare the mean opg concentration in gcf at different cal levels ( before and after treatment ) , there was a significant reduction of cal after treatment , which was proportional to a statistically significant ( p < 0.05 ) increase of opg levels in gcf as shown in table 6 . results of spearman 's rank correlation ( r ) test comparing gcf opg and cal within the groups in summary , the mean rank for the group i is the highest and it differs significantly from group ii , iii , iv , and v. further , these results indicate that gcf opg concentration decreases from periodontal health to disease . periodontitis , a chronic inflammatory disease , is characterized by increased expression of various cytokines and other inflammatory mediators resulting in extensive osteoclast formation and bone loss . these cytokines affect bone remodeling and play a vital role in both the physiological and pathological regulation of bone . opg , a key physiological inhibitor of osteoclastic bone resorption , is a glycoprotein which belongs to the tnf super family . it is a decoy receptor for rankl and inhibits cell - to - cell signaling between marrow stromal cells and precursors of osteoclasts . thus it prevents the bone resorption by rankl , which is a precursor for the production of osteoclasts . opg is expressed by various cell types like osteoblasts , osteoclastic stromal cells , t cells , b cells , chondrocytes , and follicular dendritic cells . moreover , it is also found in organs and tissues like the kidney , liver , heart , lung , spleen , thyroid , lymph nodes , thymus , brain , and placenta . it is found also in periodontal and dental tissues like gingiva and periodontal ligament and in both internal and external enamel epithelium as well as in the mesenchyme of the dental papilla during tooth development . there is a prominent expression in the cartilaginous primordia of developing maxilla , mandible , and hyoid bone . correlated opg concentration in gcf and explained its possible role in periodontal disease progression . however , till date no other studies have been documented that have compared the opg levels in gcf of subjects with healthy and diseased periodontium , and those after srp . hence , the present study was undertaken to determine the potential role of opg as a novel bone marker of periodontal disease modulator . to achieve this objective , opg levels in gcf were quantified and compared from healthy , gingivitis , slight periodontitis , and moderate - to - severe periodontitis subjects , and from moderate - to - severe periodontitis subjects after treatment . in our study , the mean concentrations of opg in gcf were found to decrease progressively from 162.47 51.171 pg/l in healthy subjects to 8.12 pg/ l in periodontitis subjects , whereas in gingivitis , mean concentration of opg was 60.50 pg/ l . the results of the present study , with respect to the general trend , are in accordance with those of mogi et al . who reported decreasing opg levels in gcf with the progression of periodontal disease . when pairwise comparison was done between healthy and gingivitis group , gingivitis and slight periodontitis group , slight periodontitis and moderate - to - severe periodontitis group , and moderate - to - severe periodontitis group and after - treatment group , healthy and slight periodontitis group , healthy and moderate - to - severe periodontitis group , and healthy and moderate - to - severe periodontitis group after treatment , the differences were statistically significant in gcf opg concentration . the mean rank obtained for moderate to severe periodontitis ( 12.50 pg/l ) was very much lower than that of healthy ( 72.50 pg/ l ) , gingivitis ( 52.44 pg/l ) , and slight periodontitis ( 44.16 pg/l ) groups . this suggests that opg levels in gcf decrease progressively from health to periodontitis . in the present study , influence of age and sex of the subjects on the opg levels was minimized by selecting the subjects within the narrow age group of 30 - 39 years and including equal number of male and female subjects in each group . further , this study comprised five groups ( healthy , gingivitis , slight periodontitis , moderate - to - severe periodontitis , and moderate - to - severe periodontitis after treatment ) as compared to the previous study ( mogi et al . 2004)where only four groups namely , healthy controls , slight periodontitis , moderate periodontitis , and severe periodontitis subjects were included , having no provision to evaluate the effect of periodontal therapy on opg levels in gcf which can further confirm the role of opg in the modulation of periodontal disease . the levels of opg reported by the earlier study were expressed as amount of opg in pg/l / site and the levels were higher than those of our study . the mean of opg levels of our study are half the concentration of opg levels of the previous study . the reasons for these variations in the levels of opg may be due to difference of study population , environmental factors , and volume of bone present during the time of gcf collection . due to these reasons , the numerical values of opg from the earlier study are not comparable with our study . the variability of opg concentration in each group could be due to different stages of the disease process at the time of collection of gcf . the levels of opg are low in the gingivitis group ( group ii ) compared to the healthy group , which could be due to near conversion of gingivitis lesion to periodontitis lesion that is not detectable clinically either by manual probing or radiography . in the slight periodontitis group , the opg concentration was less than that of healthy and gingivitis groups . the moderate - to - severe periodontitis subjects were treated by srp , and strict measures of oral hygiene were instituted . eight weeks after the treatment , the gcf samples were collected from group iv ( group v ) . the mean concentration of opg in gcf in the moderate - to - severe periodontitis group increased from 10.92 pg/l before treatment to an after - treatment level of 21.25 pg/l , respectively , which was statistically significant . recently , similar results were reported when gingival biopsies were quantified for opg mrna ( mrna : messenger rna ) and rankl in healthy and chronic periodontitis patients ( after srp ) . most of the samples of group v ( after treatment ) fall between group iii ( slight periodontitis ) and group iv , which could be due to modulation of the periodontal disease by opg where it protects the bone by preventing osteoclastogenesis accelerated by rankl . in our study , the mean mgi scores of periodontitis subjects before and after treatment were 2.27 and 1.34 , respectively , which commensurate with those of the cal levels and opg levels in gcf . in summary , our study shows that opg concentration in gcf decreases with progression of periodontal disease . further , this was accompanied with the decrease in opg levels that directly correlated with the stage of periodontal disease , that is , the amount of bone loss and cal . further , treatment aimed at arresting the progression of periodontal disease resulted in a statistically significant rise in levels of opg in gcf proportionally , confirming its active role in periodontal attachment gain . the results of the present study , therefore , provide site - specific information on changes in opg levels as a result of periodontal disease and after treatment . the source of opg in gcf seems to be from neighboring tissues including alveolar bone , gingiva , and periodontal ligament in periodontal tissues . further , the concentration of opg in gcf was increased after periodontal therapy , thus increasing the gcf opg concentration . in our study , the mean concentration of opg in group iv ( moderate - to - severe periodontitis group ) was 10.92 pg/l , lower than the concentration described in the earlier study . based on the above findings , it can be hypothesized that decreased opg levels due to progressive periodontal disease could act a as risk factor for the development of periodontal disease . however , this needs to be confirmed by conducting longitudinal , prospective studies involving larger sample size . the present study shows that the opg concentration in gcf decreases proportionally with the progression of periodontal disease , that is , gingival inflammation and cal . further , treatment aimed at arresting the progression of periodontal disease resulted in statistically significant increased levels of gcf opg proportionally , confirming its active role in periodontal attachment gain . therefore , the results of the present study provide site - specific information on changes in opg levels as a result of periodontal disease and after treatment . thus , within the limits of the present study , we can conclude that opg can be considered as a novel bone marker of the modulation of periodontal disease .

objectivesjugular foramen paraganglioma is a locally invasive , benign tumor , which grow slowly and causes various symptoms such as pulsatile tinnitus and low cranial nerve palsy . complete surgical resection is regarded as the ideal management of these tumors . the goal of this study is to identify the clinical characteristics and most effective surgical approach for jugular foramen paraganglioma.methodsretrospective analysis of 9 jugular foramen paraganglioma patients who underwent surgical resection between 1986 and 2005 was performed . clinical records were reviewed for analysis of initial clinical symptoms and signs , audiological examinations , neurological deficits , radiological features , surgical approaches , extent of resection , treatment outcomes and complications.resultsmost common initial symptom was hoarseness , followed by pulsatile tinnitus . seven out of 9 patients had at least one low cranial nerve palsy . seven patients were classified as fisch type c tumor and remaining 2 as fisch type d tumor on radiologic examination . total of 11 operations took place in 9 patients . total resection was achieved in 6 cases , when partial resection was done in 3 cases . two patients with partial resection received gamma knife radiosurgery ( gks ) , when remaining 1 case received both gks and two times of revision operation . no mortality was encountered and there were few postoperative complications.conclusionneurologic examination of low cranial nerve palsy is crucial since most patients had at least one low cranial nerve palsy . all tumors were detected in advanced stage due to slow growing nature and lack of symptom . angiography with embolization is crucial for successful tumor removal without massive bleeding . infratemporal fossa approach can be considered as a safe , satisfactory approach for removal of jugular foramen paragangliomas . in tumors with intracranial extension , combined approach is recommended in that it provides better surgical view and can maintain the compliance of the patients .

jugular foramen paragangliomas are rare , slow - growing , encapsulated , hypervascular tumors that arise from jugular foramen of temporal bone ( 1 ) . jugular foramen paraganglioma are known to occur predominantly in the age of 50 to 60 , and female to male ratio is reported to be 5:1 ( 1 , 2 ) . incidence of multiple lesion is reported to be between 25 to 50% in familial cases , compared with less than 10% in sporadic cases ( 1 ) . jugular foramen paragangliomas are locally invasive , expanding within the temporal bone via pathways of least resistance , such as air cells , vascular lumens , skull base foramina , and eustachian tube ( 3 , 4 ) . significant intracranial and extracranial extension may occur , as well as extension within the sigmoid sinus and inferior petrosal sinuses ( 3 - 5 ) . clinical course of jugular foramen paragangliomas reflects their slow growth and paucity of symptoms , which often results in a significant delay in diagnosis ( 1 , 3 , 6 ) . the most common presenting symptom is known to be pulsatile tinnitus , followed by hearing loss ( 2 , 7 ) . complete surgical resection is the ideal management of most jugular foramen paragangliomas ( 2 , 5 , 7 - 9 ) . radiation therapy can be utilized as an alternative treatment modality for certain candidates including elderly and medically inoperable patients , and is also indicated in recurred tumor after surgical resection and in residual tumors when gross total removal of extensive tumor could not be carried out ( 10 ) . gamma knife surgery is recently introduced and can be used as a primary tool in cases without significant cervical extension or in patients with recurrent tumors in intracranial area ( 10 ) . to identify the clinical characteristics and most effective surgical approach , the authors have made a retrospective analysis of the patterns of clinical presentation , surgical approaches and treatment outcomes in 9 patients with jugular foramen paraganglioma who underwent surgical treatment . the patient group consisted of 4 men and 5 women who underwent surgery by a single surgeon between 1986 and 2005 . mean age at the time of diagnosis was 40.8 yr , ranging from 26 to 60 yr . follow up period was 29 month to 264 months , with mean follow up period of 93 months . five cases occurred on the right side and remaining 4 cases on the left , and there was one case where bilateral lesion was detected . review of clinical records was performed , analyzing initial clinical symptoms and signs , audiological examinations and neurological deficits , radiological features , surgical approaches , extent of resection , treatment outcomes and postoperative complications . the extent of jugular foramen paraganglioma was classified according to fisch classification , based on radiological findings . most common initial symptom at the time of first outpatient visit was hoarseness which was observed in 4 patients , followed by pulsatile tinnitus which was noted in 3 cases ( table 1 ) . sudden sensorineuronal hearing loss in 2 patients , facial palsy in 2 patients and neck mass in 1 patient were also reported . all patients except for 2 cases had at least one low cranial nerve palsy on evaluation . most common neurologic deficit on presentation was 10th nerve palsy , followed by 9th nerve , 12th nerve , and 7th nerve in order . the extent of jugular foramen paragangliomas were classified according to fisch classification with radiologic findings of mri . seven patients were classified as type c ( fig . 1 ) and remainnig 2 patients were type d ( fig . size of the tumor varied from as small as 1.51 cm to as large as 4.59 cm . on angiography , all tumors had at least one main feeding vessel . ascending pharyngeal artery was the most common feeding vessel , and successful embolization of the feeding vessel was achieved in all cases ( fig . complete resection was achieved in 6 patients , and all of them is under regular outpatient follow - up without any sign of recurrence . all type d tumors resulted in partial resection and 1 out of 7 type c tumors was partially resected . the residual tumor in the 2 cases of type d jugular foramen gangliomas was the portion of intracranial extension . in one type d tumor case ( case 5 ) , 1 cm sized residual tumor was suspected in postoperative mri after first operation in 1999 . the patient received gamma knife radiosurgery ( gks ) on the remnant tumor and was continuously observed with mri at the outpatient . however , the patient had to undergo revision operation in cooperation with neurosurgery department in 2006 because the tumor mass grew as large as 4 cm . tumor still remained after the second operation and revision operation by neurosurgery department was performed in 2007 , still resulting in small portion of residual tumor . the remaining tumor is under observation currently . in the remaining two partial resection cases , however , the patients refused to undergo the burden of major surgery twice after the first operation ( infratemporal approach ) , and gamma knife surgery had to be performed as an alternative to planned staged operation by neurosurgery department . tumor was completely shrunken in one case , and tumor was stabilized without growing in the other case . no mortality was encountered during the follow - up period , ranging from 17 to 275 months ( mean , 71 months ) . in all 4 cases , patient showed h - b grade iv palsy which returned to h - b grade ii palsy in a few months . since infratemporal fossa approach inevitably encounters facial palsy due to transposition of facial nerve , facial palsy of grade better than h - b grade ii three patients received arytenoid adduction , 2 cases due to unilateral vocal cord palsy , and one case due to decreased vocal cord mobility . in other case , left vocal cord palsy developed , but the patient did not undergo arytenoid adduction since symptom due to vocal cord palsy was not prominent . most common initial symptom at the time of first outpatient visit was hoarseness which was observed in 4 patients , followed by pulsatile tinnitus which was noted in 3 cases ( table 1 ) . sudden sensorineuronal hearing loss in 2 patients , facial palsy in 2 patients and neck mass in 1 patient were also reported . all patients except for 2 cases had at least one low cranial nerve palsy on evaluation . most common neurologic deficit on presentation was 10th nerve palsy , followed by 9th nerve , 12th nerve , and 7th nerve in order . the extent of jugular foramen paragangliomas were classified according to fisch classification with radiologic findings of mri . seven patients were classified as type c ( fig . 1 ) and remainnig 2 patients were type d ( fig . size of the tumor varied from as small as 1.51 cm to as large as 4.59 cm . on angiography , all tumors had at least one main feeding vessel . ascending pharyngeal artery was the most common feeding vessel , and successful embolization of the feeding vessel was achieved in all cases ( fig . complete resection was achieved in 6 patients , and all of them is under regular outpatient follow - up without any sign of recurrence . all type d tumors resulted in partial resection and 1 out of 7 type c tumors was partially resected . the residual tumor in the 2 cases of type d jugular foramen gangliomas was the portion of intracranial extension . in one type d tumor case ( case 5 ) , 1 cm sized residual tumor was suspected in postoperative mri after first operation in 1999 . the patient received gamma knife radiosurgery ( gks ) on the remnant tumor and was continuously observed with mri at the outpatient . however , the patient had to undergo revision operation in cooperation with neurosurgery department in 2006 because the tumor mass grew as large as 4 cm . tumor still remained after the second operation and revision operation by neurosurgery department was performed in 2007 , still resulting in small portion of residual tumor . the remaining tumor is under observation currently . in the remaining two partial resection cases , however , the patients refused to undergo the burden of major surgery twice after the first operation ( infratemporal approach ) , and gamma knife surgery had to be performed as an alternative to planned staged operation by neurosurgery department . tumor was completely shrunken in one case , and tumor was stabilized without growing in the other case . no mortality was encountered during the follow - up period , ranging from 17 to 275 months ( mean , 71 months ) . in all 4 cases , patient showed h - b grade iv palsy which returned to h - b grade ii palsy in a few months . since infratemporal fossa approach inevitably encounters facial palsy due to transposition of facial nerve , facial palsy of grade better than h - b grade ii three patients received arytenoid adduction , 2 cases due to unilateral vocal cord palsy , and one case due to decreased vocal cord mobility . in other case , left vocal cord palsy developed , but the patient did not undergo arytenoid adduction since symptom due to vocal cord palsy was not prominent . our collected data showed some differences from the previous reports on jugular foramen paraganglioma . in previous reports , jugular foramen paraganglioma is known to occur predominantly in the age of 50 to 60 , and female to male ratio is reported to be 5:1 ( 1 , 2 ) . however , the female to male ratio was 5:4 , and tumors were detected at the mean age of 40.8 yr in our data , even though the age of detection was distributed widely , not being focused to certain age group . our data showed no significant preference to certain sex , age and site , in terms of incidence . most common initial presenting symptom at the time of first visit to the outpatient was hoarseness , whereas the most common presenting symptom is known to be pulsatile tinnitus , followed by hearing loss in the previous reports ( 2 , 7 ) . lower cranial nerve dysfunction is relatively common with glomus jugulare tumors and includes dysphagia , hoarseness , aspiration , tongue paralysis , shoulder drop , and voice weakness ( 3 , 5 , 7 ) . our result was consistent with that , and 7 out of 9 patients showed at least one low cranial nerve palsy , emphasizing the importance of neurologic evaluation of low cranial palsy at the time of first visit . emphasis should also be given on the fact that patients without pulsatile tinnitus should not be excluded from the suspicion of jugular foramen paraganglioma . facial nerve paralysis is known to occur less commonly , but it signals advanced disease and is related with poor facial nerve prognosis outcome ( 3 , 5 , 7 ) . facial palsy was present in 2 cases in our data and both cases were advanced jugular foramen paragangliomas . facial nerve function was not restored from the initial facial nerve palsy after the operation in both cases . on imaging evaluation with mri , 7 patients were type c tumors and remaining 2 cases were type d tumors . it correlates with the fact that jugular foramen paragangliomas are slow growing and lack symptoms which results in the late diagnosis of the tumor ( 1 , 3 , 6 ) . successful embolization and surgical removal of tumor without massive bleeding were achieved in all cases . ascending pharyngeal artery was the most common feeding vessel , as reported previously ( 11 ) . since the tumor is hypervascular in nature , angiography is crucial to identify main feeding vessels and embolize them prior to surgery which decreases the chance of massive bleeding intraoperatively ( 11 ) . complete surgical resection is the ideal management of most jugular foramen paragangliomas ( 2 , 5 , 7 - 9 ) . in our cases , 6 out of 7 type c tumors were resected completely and are under observation without any sign of recurrence . all type d tumors resulted in partial resection and 1 out of 7 type c tumors was partially resected . gks after partial resection of the tumor was reported to be effective ( 10 ) , but recurrence was observed in 1 out of 3 cases in our data . we recommend that combined approach in cooperation with the neurosurgery department should be implemented in the first place in cases of jugular foramen ganglioma with intracranial extension , rather than planning a staged operation . showed that a single - stage resection and reconstruction offered the greatest likelihood of complete tumor removal while preserving local tissue for use in reconstruction ( 12 ) . combined approach can include infratemporal fossa , suboccipital , middle fossa , anterior craniofacial , translabrynthine approach , and more . these approaches can be combined depending on the location of the tumor ( 12 , 13 ) . combined approach can expose the tumor better than single approach since better operative view of the tumor can be obtained from two different angles , which should result in better chance of complete removal of the tumor ( 13 ) . the widened view also prevents excessive effort to remove unvisualized portion of the tumor , which might cause injuries to the nerves . there is a problem to seal a larger defect in combined approach compared with a single approach , but advances in reconstruction technique of the defect has decreased concerns to this problem ( 12 , 13 ) . combined approach is also much better to maintain compliance of the patient for the treatment . in our cases , all patients in whom staged operation was planned refused to undergo the secondary operation due to decreased compliance after the first operation . in our cases , infratemporal approach was sufficient in complete resection of the tumor in most cases when intracranial extension was not present , with the aid of embolization . no mortality was encountered and permanent postoperative complications occurred in few cases , as observed in the results . with skilled surgical technique , infratemporal fossa approach can be considered as a safe and effective approach for removal of jugular foramen paraganglioma without intracranial extension . low cranial involvement was present in most of the cases , suggesting that neurologic examination of the low cranial nerve is crucial . in the surgical aspect , it can be concluded that infratemporal fossa approach provides safe , satisfactory way to remove jugular foramen paraganglioma , but combined approaches is strongly recommended instead of staged operation when treating jugular foramen paraganglioma with intracranial extension .

aims : to study the demographic factors associated with alcohol dependence syndrome so that the problems of alcohol related co morbidities can be prevented with appropriate preventive measures.materials and methods : the study was conducted in de - addiction clinic of the department of psychiatry , mamata medical college , khammam , andhra pradesh from july 2008 to february 2009 . patient who fulfills criteria for alcohol dependence , according to diagnostic and statistical manual of mental disorders , fourth edition were included.results:mean age ( standard deviation ) at first drink was 18.93 ( 3.81 ) years and at onset of alcohol dependence was 28.28 ( 6.55 ) years . the most common reason being given by the patients was financial strain ( 70% of the patients ) due to alcohol use and its consequences . educational qualification of 12th standard or above was seen only in 7.5% . alcohol dependence syndrome was more common in unemployed , unskilled and semi - skilled patients . majority of patients ( 80% ) belonged to lower socio - economic class.conclusion:alcohol dependence syndrome and its related co morbidities can be minimized to a great extent if the educational and socio - economic standards are improved in countries like india where there is increase in alcohol consumption as a life style choice .

the pattern of drinking in india has undergone a change from occasional and ritualistic use to being a social event . these developments have raised concerns about the health and the social consequences of excessive drinking leading to dependence . in the last decade there is a rapid increase in the number of city bars and nightclubs in india and as a result an undocumented rise in alcohol abuse amongst all the sections of the society . the percentage of the drinking population aged less than 21 years has increased from 2% to more than 14% in the past 15 years , according to studies in the southern state of kerala by alcohol and drugs information center india , a non - governmental organization . what is of particular concern and an important indicator of health risks is that the signature pattern of alcohol consumption in india is frequent and heavy drinking . more than half of all drinkers fall into the criteria for hazardous drinking , which is characterized by bingeing and solitary consumption to the point of intoxication . the present study is carried out to look for the demographic factors associated with alcohol dependence syndrome so that the problems of alcohol related co morbidities can be prevented with appropriate preventive measures . the study was conducted in de - addiction clinic of the department of psychiatry , mamata medical college and associated mamata general hospital , khammam , andhra pradesh during the period from july 2008 to february 2009 . all the subjects fulfilling the inclusion and exclusion criteria during the study period were included in this study . all male subjects presenting to the de - addiction clinic of the mamata general hospital with alcohol related problems were potential candidates for this study and they are enrolled into the study if they fulfill the following inclusion criteria : patient of age 18 years and abovepatient who fulfills criteria for alcohol dependence , according to diagnostic and statistical manual of mental disorders , fourth edition ( dsm - iv ) . patient of age 18 years and above patient who fulfills criteria for alcohol dependence , according to diagnostic and statistical manual of mental disorders , fourth edition ( dsm - iv ) . subjects with any of the following will not be included in the study : concurrent presence of other substance dependence , according to dsm - iv , other than nicotinepresence of any significant illness requiring intensive medical / surgical management . concurrent presence of other substance dependence , according to dsm - iv , other than nicotine presence of any significant illness requiring intensive medical / surgical management . patients fulfilling the selection criteria were admitted after obtaining informed consent and demographic details , history , general physical examination and mental status examination were recorded on a semi - structured proforma designed for the study . all male subjects presenting to the de - addiction clinic of the mamata general hospital with alcohol related problems were potential candidates for this study and they are enrolled into the study if they fulfill the following inclusion criteria : patient of age 18 years and abovepatient who fulfills criteria for alcohol dependence , according to diagnostic and statistical manual of mental disorders , fourth edition ( dsm - iv ) . patient of age 18 years and above patient who fulfills criteria for alcohol dependence , according to diagnostic and statistical manual of mental disorders , fourth edition ( dsm - iv ) . subjects with any of the following will not be included in the study : concurrent presence of other substance dependence , according to dsm - iv , other than nicotinepresence of any significant illness requiring intensive medical / surgical management . concurrent presence of other substance dependence , according to dsm - iv , other than nicotine presence of any significant illness requiring intensive medical / surgical management . patients fulfilling the selection criteria were admitted after obtaining informed consent and demographic details , history , general physical examination and mental status examination were recorded on a semi - structured proforma designed for the study . a total of 68 male patients were registered at the de - addiction clinic , at the department of psychiatry , mmc and mamata general hospital , khammam between the period july 1 , 2008 and february 28 2009 . whereas a total of 63 patients met the dsm - iv criteria for alcohol dependence , 7 were excluded from the study as they met criteria for another drug dependence . the remaining 56 patients were each offered admission for further management , but 16 patients refused / did not come for admission . the mean age at presentation in the study was 37.2 ( 8.51 ) years . mean age at presentation in various studies with similar design this study found that 62% of patients were married at time of presentation [ figure 1 ] . in most western studies , the marital status of the patient has been most commonly found to be being separated or divorced , ranging between 43% and 60% respectively . one large study in 2713 alcohol dependent patients however , reports higher rate of married patients than controls ( 57% ) . the differences are possibly due to cultural differences . sample in the current study came from a predominantly rural population , mostly from nuclear families . this reflects the population being catered to by the hospital where this study took place ; an urban center with huge rural catchment area 32 ( 80% ) [ table 1 ] patients had education less than high school level . most of the studies have shown that most of the patients in similar clinical settings had education less than high school [ table 2 ] . showing the marital status of the individuals type and background of family the educational status of the individuals rate for various forms of employment was 85% , involving mostly unskilled or skilled work . previous studies have reported an equal percentage of patients who are unemployed or are gainfully employed . the current study had higher rates of patients who were employed , but mostly involved in work that did not require much education and hence , comparison becomes difficult [ table 3 ] . the majority of patients in the study belonged to the lower middle and middle socio - economic class . other studies too have reported their patient population as belonging to lower middle to lower class [ table 4 ] . mean age at which first drink of alcohol ( in any form or amount ) was taken is 18.9 years [ table 5 ] . the mean age at getting intoxicated by the alcohol for the first time ( i.e. , feeling the effects of alcohol , or taking an equivalent of 80 - 90 ml or more of alcohol in one setting or within a brief period of approximately 1 - 2 h ) was 19.8 years . a study among in - patients reported the mean age at first consumption to be 15.4 years and at regular consumption to be 23.1 years . the collaborative study of the genetics alcoholism ( coga ) group found the mean age at onset of alcoholism was 25 years [ table 5 ] . the mean age at onset alcohol abuse was 24 years and at onset of alcohol dependence ( according to dsm - iv ) was 28.3 years . a study by powell et al . , had found mean age at onset of drinking was 17.3 years , while age at problem - level drinking was 30.4 years . majority of patients had presence of withdrawal symptoms ( 95% ) and tolerance ( 95.5% ) [ table 6 ] . dsm - iv indicates that diagnoses of substance dependence should be further characterized with regard to the presence of a physiological component . coga study found that such a distinction predicated more physiological complications and more alcohol - related emotional / psychiatric symptoms such as depression and anxiety . withdrawal symptoms and tolerance in this study , the patients and key informants were asked to enumerate reasons for seeking current treatment [ tables 7 and 8 ] . based on the compilation of various reasons , these could be grouped into certain categories . on analysis , financial strain due to alcohol use was most commonly attributed for current treatment seeking by both patients ( 70% ) and key informants ( 92.5% ) , though the patients mostly denied it as their first reason . this was followed by presence of family conflicts and concern about physical health ( 65% each ) , followed by seeking treatment due to social pressure ( 35% ) , experiencing withdrawal symptoms ( 25% ) , or psychological reasons ( 22.5% feelings of guilt , low mood , etc . ) . most of the patients had more than 2 reasons for seeking treatment and this was comparable to reasons provided by the key informats . this was a unique effort in this study to incorporate such clinical parameters into the assessment , as it seems to yield similar severity status . studies reported similarly reasons for admission to current treatment in which decision by the patient was the most common reason ( 73 - 76% of patients ) , while other reasons included medical condition ( 12% ) and employer pressure ( 8% ) but stopped short of further analysis . reasons for seeking treatment informants reasons for seeking treatment mean age at first drink was 18.9 years and age at onset of alcohol dependence was 28.3 years . the alcohol dependence syndrome was more common in low socio - economic class and people with education up to high school level . measures to improve the education and socio - economic standards in the rural areas are urgently needed to control the alcohol abuse and related co morbidities .

summarydespite the large evolutionary distances , metazoan species show remarkable commonalities , which has helped establish fly and worm as model organisms for human biology1,2 . although studies of individual elements and factors have explored similarities in gene regulation , a large - scale comparative analysis of basic principles of transcriptional regulatory features is lacking . we mapped the genome - wide binding locations of 165 human , 93 worm , and 52 fly transcription - regulatory factors ( rfs ) generating a total of 1,019 data sets from diverse cell - types , developmental stages , or conditions in the three species , of which 498 ( 48.9% ) are presented here for the first time . we find that structural properties of regulatory networks are remarkably conserved and that orthologous rf families recognize similar binding motifs in vivo and show some similar co - associations . our results suggest that gene - regulatory properties previously observed for individual factors are general principles of metazoan regulation that are remarkably well - preserved despite extensive functional divergence of individual network connections . the comparative maps of regulatory circuitry provided here will drive an improved understanding in the regulatory underpinnings of model organism biology and how these relate to human biology , development , and disease .

data sets described here can be obtained from the encode project website at www.encodeproject.org / comparative / regulation/. all data sets were processed using a uniform processing pipeline with identical alignment and filtering criteria and standardized idr peak calling using spp ( human + worm ) and macs2 ( fly ) . ( a ) 32 tf gene families with a binding dataset for at least two species ( names abbreviated ) . cross enrichment indicates the enrichment of motifs from one species in the datasets of another . for 13 families , we observed no cross enrichment ( red ) . for 7 families ( blue ) we observed cross enrichment and for an additional 12 ( green ) we also had matching motifs . for two cases marked by an asterisk a known fly motif matches the human motif but no worm motif matches . we discovered a motif in worm datasets that match literature derived known motifs from human and fly . ( c ) all three motifs are highly similar and enriched in human prdm1 and worm blmp-1 datasets . enrichments in each box are the fraction of motif instances that are inside the bound regions and dividing that by the fraction of shuffled motif instances . additional motifs known and discovered for these and other datasets are included in supplementary information . ( a ) fly - worm stage alignment of expression using all fly - worm orthologs . ( d ) fly - worm stage alignment by using proximal genes to chipd tf binding sites . the stage - mapped data exhibit two sets of collinear patterns between the two species ( distinct diagonals ) . in the bottom diagonal , expression from worm embryos and larvae are matched with fly embryos and larvae , respectively ; worm adults are matched with fly early embryos and fly female adults , possibly due to the orthologous gene expression in eggs of both species ; worm dauers are matched with fly late embryo to l1 and l3 stages , which is similar to the position of dauer stages in the worm lifecycle ( between worm l1 and l4 stages ) . in the upper diagonal , worm middle embryos are matched with fly l1 stage ; worm late embryos are matched with fly prepupae and pupae stages ; worm l4 male larvae are matched with fly male adults . this collinear pattern may be attributable to fly genes with two - mode expression profiles and many - to - one fly - worm orthologous gene pairs . for more details a comparison of go enrichment of orthologous tf pairs for all contexts in ( a ) human vs worm , ( b ) human vs. fly , and ( c ) worm vs. fly is shown . plus signs mark orthologous tf pairs with white pluses indicating the most significant enrichment for an ortholog pair . ( d ) orthologous factors are more enriched for matching go terms than non - orthologous factors . red dash line represents hot threshold and black dashed line represent an enrichment of 1x . black line represents best fitting line to the scatter plot ( r = 0.0045 ) ( b ) a scatterplot of density ( number of tf peaks per kb ) rather than total number of peaks in a region shows a similar trend . ( c ) barplot of fraction of regions with high igg enrichment for hot and non - hot ( rgb ) regions using the same threshold ( 1.5x ) as teytelman et al . ( d ) the fraction of hot ( red ) and non- hot ( blue ) regions with high igg enrichment is plotted as a function of threshold . black line represents no enrichment ( igg / input = 1x ) and grey dashed line represents the enrichment cutoff ( 1.5x ) used in ( b ) and in teytelman et al . ( e ) comparison of igg ( igg / input ) and rna pol ii enrichment ( rna polii / input ) shows a different trend from teytelman et al . ( e ) nearly all ( 99.967% ) of our uniformly processed rna polii binding sites have ip / input rations > 2x , with a median enrichment of ~20x . ( a ) to identify hot region for each context , we first analyzed the number and size distribution of target binding regions ( in which factor binding sites are concentrated ) . for each target case simulation , we randomly select an equivalent number of random binding regions with a matched size distribution . next , for each factor assayed ( in the target case ) , we evaluated the number and size of observed binding sites , and simulated an equivalent number and size distribution of target binding sites , restricting their placement to the simulated binding regions . we collapsed simulated binding sites from all factors into binding regions , verifying that these cluster into a similar number of simulated binding regions as the target binding regions . we identify regions at a 5% ( hot ) and 1% ( xot ) occupancy threshold based on this simulated data . ( b ) binding of regulatory factors covers different fractions of the genomes of fly , human , and worm . coverage is shown for constitutively hot regions ( chot red ) , hot regions ( yellow ) , and non - hot regions ( rgb green ) . ( a ) histone marks for hot regions ( represented by points and smoothed to show density ) at proximal and ( b ) distal sites show similar trends of histone mark enrichment in their flanking regions . enhancer calls for a specific developmental stage ( c , e ) or cell type ( d ) ( labeled over each set of bar graphs ) match hot regions from that cell type and not hot regions from another cell type . each set of six bar graphs represents the same set of hot regions called constitutively hot or specific to each of the five cell types . constitutive hot ( chot ) regions are significantly enriched at promoters with the remaining regions overlapping enhancer regions . the number of ffls in a stage is normalized by the number of tfs in the corresponding stage - specific network . though the sets of tfs may differ , the number of tfs in each stage stays roughly the same . the pairwise co - association strengths between orthologous tfs are shown for human - worm orthologs ( a , b ) and human - fly orthologs ( c , d ) . for each pair of species - specific orthologs across multiple samples , the co - association strength , measured as the fraction of significant co - binding events between experiments , is shown ( intervalstats ) . the co - association strength of human - worm orthologs in human ( x - axis ) is plotted against the co - association strength in worm ( y - axis ) . lines depict 1 ( solid ) and 1.5 ( dashed ) standard deviations from the mean score . factors in blue represent enrichments due to paralogous tfs in human that tend to be highly co - associated . co - association strength in human ( x - axis ) is plotted against co - association strength in fly ( y - axis ) . for tf orthologs assayed in multiple developmental stages / cell - lines , the maximal co - association between contexts was selected for the comparative analyses ( e , f ) .

immunohistochemistry has recently been validated for the detection of the brafv600e mutation across a range of tumor types . in colorectal carcinoma , the presence of the brafv600e mutation can be used to virtually exclude lynch syndrome in mismatch repair - deficient tumors . in mismatch repair - proficient tumors , brafv600e mutation assessed by molecular methods has been proposed as a poor prognostic factor . we investigated whether combined brafv600e and mismatch repair status assessment by immunohistochemistry alone can be used as a prognostic marker in the routine clinical setting . we performed immunohistochemistry for brafv600e , mlh1 , pms2 , msh2 , and msh6 on 1426 consecutive unselected colorectal carcinomas . ninety - one ( 6.4% ) carcinomas were mismatch repair - proficient and brafv600e mutant , and these tumors demonstrated a significantly worse 5-year survival of 49.7% compared with mismatch repair - proficient braf wild type ( 74.1% of tumors , 65.4% survival ) , mismatch repair - deficient brafv600e mutant ( 12.9% of tumors , 70.1% survival ) , and mismatch repair - deficient braf wild type ( 6.6% of tumors , 73.6% survival ) . the poor survival was confirmed by univariate analysis ( p<0.01 ) but fell away in multivariate analysis ( p=0.68 ) because of the strong effect of tumor stage and age on overall survival . we conclude that in addition to its utility in screening for lynch syndrome , reflex brafv600e and mismatch repair assessment by immunohistochemistry can be used as a powerful predictor of all - cause survival .

we searched the pathology database of the royal north shore hospital , sydney , australia , for all colorectal carcinomas undergoing surgical resection during the calendar years 20042009 . during this period , this center performed centralized pathological testing for two quaternary referral hospitals with dedicated colorectal surgery units and four community hospitals . therefore , this patient cohort represents a true snapshot of colorectal carcinoma cases encountered in the community as a whole . tissue microarrays containing duplicate 1 mm cores were created and immunohistochemistry for the four mismatch repair proteins ( mlh1 , msh2 , pms2 , and msh6 ) and brafv600e ( using clone ve1 , springbioscience , pleasonton , ca ) were performed using previously described methods . for brafv600e mutation - specific immunohistochemistry , slides were stained using the leica bondiii autostainer ( leica microsystems , mount waverley , vic , australia ) used according to the manufacturer 's protocol . the slides were dewaxed in bond dewax solution ( ar9222 , leica microsystems ) and hydrated in bond wash solution ( ar9590 , leica microsystems ) . heat - induced epitope retrieval was performed for 60 min in the manufacturer 's alkaline retrieval solution er2 ( vbs part no : ar9640 , leica microsystems ) . slides were then incubated with the primary antibody at a dilution of 1 in 80 for 30 min at room temperature . antibody detection was performed using the biotin - free bond polymer defined detection system ( ds9713 , leica microsystems ) according to the manufacturer 's protocol . if the tissue microarray staining was not clearly interpretable ( eg , if there were no good internal controls for the mismatch repair markers ) , it was repeated on whole sections . follow - up was obtained by the examination of hospital medical records , the records from surgeons ' private rooms , and archival public death notices and obituaries in the state of new south wales , australia . patients were followed up until death or their last date of follow - up not > 7 years after definitive surgery . the survival for each of the four immunohistochemical phenotypes was determined as a function of cumulative survival over time ( kaplan meier method ) . multivariate cox regression was employed to explore the effect of mismatch repair / braf immunohistochemistry phenotype on survival , using a model that included gender , age at diagnosis , tumor anatomic location , tumor histologic grade , presence or absence of lymphovascular space invasion , peritumoral lymphocyte reaction status , and american joint committee on cancer / tnm seventh edition tumor stage . this study was approved by the northern sydney local health district human research ethics committee under protocol 1201 - 035 m . briefly , the mean age was 74 years ( range 17100 years ) and 52.1% were females . one thousand one hundred and forty - eight ( 80.5% ) were mismatch repair - proficient , comprising 1057 ( 74.1% ) mismatch repair - proficient braf wild type and 91 ( 6.4% ) mismatch repair - proficient / brafv600e mutant . one hundred and eighty - four cases ( 12.9% ) were mismatch repair - deficient / brafv600e mutant and 94 ( 6.6% ) mismatch repair - deficient / braf wild type . during follow - up , ( mean 5.29 years , 75th centile 3.21 years ) , 353 patients died . survival curves correlating immunohistochemical staining pattern and survival by both kaplan meier and cox regression methods are presented in figure 1 along with photomicrographs of representative staining patterns . the 5-year survivals progressively deteriorated from mismatch repair - deficient / braf wild type ( 73.6% ) to mismatch repair - deficient / brafv600e mutant ( 70.1% ) , to mismatch repair - proficient / braf wild type ( 65.4% ) to mismatch repair - proficient / brafv600e mutant ( 49.7% ) . pairwise comparisons with mismatch repair - proficient / braf wild - type colorectal carcinomas as the baseline group in univariate cox regression modeling revealed overlapping 5-year survival figures for all tumor groups , except mismatch repair - proficient / brafv600e mutant tumors , which showed a statistically significant worse outcome hazard ratio of death 1.79 ( 95% ci=1.242.60 ) , p<0.01 . in multivariate analysis , the poor prognosis of mismatch repair - proficient / brafv600e mutant tumors was negated ( hazard ratio of 1.10 ( 95% ci=0.691.76 ) , p=0.68 ) by the dominant effect of stage and age on overall survival . however , the better prognosis of mismatch repair - deficient / brafv600e mutant tumors became significant ( hazard ratio of 0.57 ( 95% ci=0.35093 ) , p=0.03 ) when compared with the baseline group of mismatch repair - proficient / braf wild - type tumors ( table 1 ) . the use of molecular markers to predict outcome in colorectal carcinoma , particularly mismatch repair deficiency / microsatellite instability and braf mutation , has been an area of active research . briefly , most studies have indicated that mismatch repair deficiency is associated with a good outcome . in contrast , the presence of braf mutation is usually found to be a marker of poor prognosis . although some studies have found that braf mutation does not predict outcome in an unselected population , this discrepancy appears to be because of the strong association between braf mutation ( a poor prognostic factor ) with mismatch repair deficiency ( a good prognostic factor ) through the somatic hypermethylation pathway . therefore , recently several groups have used the combination of mmr and braf mutation status as determined by molecular means to predict outcome in colorectal cancer . using this approach , mismatch repair - deficient / braf wild - type colorectal carcinomas have been consistently found to have a good prognosis , whereas mismatch repair - proficient / brafv600e mutant colorectal carcinomas have emerged as a poor prognostic group in most but not all studies . this combined mismatch repair / braf prognostic algorithm was tested recently by lochhead et al who used molecular techniques to determine microsatellite instability and braf mutation status in 1253 colorectal carcinoma patients . compared with the majority of tumors that were mismatch repair - proficient / braf wild type , mismatch repair - proficient / braf mutant colorectal carcinomas demonstrated a poor prognosis ( hazard ratio of colon cancer - specific mortality 1.6 ( 95% ci=0.122.28 ) ) . mismatch repair - deficient / braf mutant colorectal carcinomas demonstrated a good prognosis with a hazard ratio of 0.48 ( 95% ci=0.270.87 ) and mismatch repair - deficient / braf wild type demonstrated a very good prognosis with a hazard ratio of 0.25 ( 95% ci=0.120.52 ) . the results of our study , although based on all cause rather than cancer - specific survival , are essentially confirmatory of lochhead et al , with the significant advantage that we did not use any molecular techniques , only immunohistochemistry an approach that is readily available in virtually any diagnostic surgical pathology laboratory . in centers where universal lynch syndrome screening is already undertaken with mismatch repair deficiency immunohistochemistry for mlh1 , pms2 , msh2 , and msh6 , it would simply be a matter of performing immunohistochemistry for five markers rather than four , with estimated additional disposable costs of < us$10 and minimal extra labor costs . we note that the very poor prognosis of mismatch repair - proficient / braf mutant tumors found in univariate analysis ( p<0.01 ) fell away in multivariate analysis because of the dominant effect of stage and age on overall survival . however , given the ease with which braf status can be determined in conjunction with mismatch repair deficiency in the routine clinical setting , and the established indication for combined mismatch repair deficiency and braf testing to triage formal genetic testing for lynch syndrome , our study makes an argument for its potential use as a prognostic marker in all colorectal cancers . to date , four of the five independent studies investigating brafv600e mutation - specific immunohistochemistry in colorectal carcinoma have determined that it is highly sensitive and specific for the presence of the brafv600e mutation with only one study suggesting limited utility . it would be reasonable to conclude that local factors such as tissue processing techniques and staining methods can affect the performance of the antibody , but most laboratories including our own have found it to be a robust and reliable marker . however , we do caution that introduction of braf immunohistochemistry should only be performed with the appropriate quality control measures , including the use of controls and the validation of the accuracy of the testing in individual laboratories . previous studies of brafv600e mutation - specific immunohistochemistry in colorectal carcinoma have concentrated on its utility in triaging formal genetic testing for lynch syndrome in microsatellite - unstable tumors . other studies have concentrated on the prognostic predictive power of combined braf and mismatch repair deficiency assessment when determined by molecular means . this is the first study to demonstrate that braf determination by immunohistochemistry alone , when interpreted in conjunction with mismatch repair deficiency status , also identifies subgroups of colorectal carcinomas with different overall survivals most significantly the poor prognostic group of mismatch repair - proficient / brafv600e mutant tumors . in summary , our results suggest that the addition of brafv600e immunohistochemistry to mismatch repair deficiency immunohistochemistry identifies distinct prognostic subgroups of colorectal carcinomas , including the poor prognostic group of mismatch repair - proficient / brafv600e mutant tumors . if our results are confirmed in other large independent cohorts , a strong argument could be made to perform routine brafv600e immunohistochemistry at the same time as mismatch repair deficiency assessment on all colorectal carcinomas , not just to facilitate universal screening for lynch syndrome in mismatch repair - deficient tumors but also to identify the poor prognosis mismatch repair - proficient / brafv600e mutant group .

determining radioactivity levels in foodstuffs is of great importance for the protection of human health . in addition , the literature includes few studies related to this subject in turkey . in this study , gamma spectroscopic system was used in order to measure 226ra , 232th , 40k and 137cs activity concentrations in vegetables and fruits produced in elaz region . the average activity concentrations in vegetables was calculated as 0.64 0.26 bq kg1 for 226ra , 0.65 0.14 bq kg1 for 232th , 13.98 1.22 bq kg1 for 40k , and 0.54 0.04 bq kg1 for 137cs . the average activity concentrations in fruits were 1.52 0.34 , 0.98 0.23 , 18.66 1.13 and 0.59 0.16 bq kg1 , respectively for 226ra , 232th , 40k and 137cs . total committed effective dose value was determined as 20 and 30.55 sv y1 , respectively for vegetables and fruits . the findings were compared with previous data reported for turkey and other regions of the world .

natural sources of radioactivity in the environment are called naturally occurring radioactive materials , and are categorized as being of terrestrial or cosmic origin . humans are exposed to both internal and external radiation from these natural sources . internal exposure occurs through the intake of terrestrial radionuclides through inhalation or ingestion . inhalation exposure dose results from the existence of dust particles in air , including radionuclides from u and th decay series . the biggest contribution to inhalation exposure comes from short half - life decay products of radon . ingestion exposure dose mostly results from u and th series radionuclides and k in drinking water and foodstuff . in addition , cs is the most important fission product released to the environment as a result of nuclear activities , because this radionuclide rapidly passes to foodstuffs and creates a dose effect . the aim of this study is to determine the exposure dose of ra , th , k and cs radionuclide concentrations in fruits and vegetables produced in the elaz region of turkey , which are frequently consumed by local residents . the significance of the study is that it is the first study to determine the background radiation levels in such food products in this region and will provide data for future studies and in case of a nuclear accident ( as in chernobyl nuclear accident ) or nuclear fallout , to determine level of contamination . the province of elaz is located in the eastern anatolian region , between longitude 38304021e and latitude 38173911n . its surface area is 9,151 km and the average altitude is 1,067 m. the region is divided into 11 administrative regions , with a total population of 540,000 ( fig . 1 ) . approximately 50 % of the province consists of grasslands , 28 % is agricultural land , 12 % forest , and 10 % is dams and lakes . a continental climate prevails ; winters are cold and snowy , and summers are hot and arid . the province is rich in mineral resources , and mining activities include copper , fluoride , chalcopyrite , zinc , lead , chrome , manganese , molybdenum , iron and wolfram .fig . samples of fruits and vegetables produced and frequently consumed in the region were provided from a public market . any soil or foreign materials on the samples were removed so that they were suitable for consumption , divided into small pieces , and washed under distilled water . they were kept at room temperature for 3 months without allowing any contamination and then totally oven - dried at 105 c . the temperature of the oven was increased to 250 c and was continued until the samples were reduced to ash . the ashed samples were then homogenized and transferred into a plastic container ( 5 cm height 5 cm diameter ) . finally , the samples were sealed and stored for a period of about 1 month before counting , in order to allow equilibrium between ra and its short - lived decay products . the activity concentrations of ra , th , k and cs radionuclides in vegetable and fruit samples were determined using a gamma spectroscopic system , comprising a 2 2 nai(tl ) well - type detector and a detector surrounded by a cylindrical lead shield ( thickness , diameter and length approximately 3.5 , 13.7 and 15.5 cm , respectively ) . energy calibration of detector was performed by using co ( 37 kbq ) and ra ( 370 kbq ) point sources . ra , th , k and cs activity concentrations in vegetable and fruit were based on the detection of 609.3 , 583 , 1461 and 662 kev energy gamma rays transmitted by bi , tl , k and cs , respectively . ( 1)1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a({\rm{bq}}\,{\rm{kg}}^ { { - 1 } } ) = \frac{c}{{m_{{\rm{s } } } \varepsilon p_{\gamma } } } $ $ \end{document}where c is the gamma ray count ( number per second ) , is the detector efficiency of the specific gamma ray , p is the absolute transition probability of gamma decay and ms is the mass of the sample ( kg ) . ingestion dose occurring through the intake of radionuclides depends on the consumption rate of foodstuff and the concentration of the radionuclide involved . ( 2 ) [ 3 , 13 , 14]2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ h_{\text{t , r } } = \mathop \sum \nolimits \left ( { u^{i } c_{\rm{r}}^{i } } \right)g_{\text{t , r } } $ $ \end{document}where i is foodstuff group , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ u^{\it i } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$c_{\rm{r}}^{i } $ $ \end{document } are annual consumption rate ( kg ) and radionuclide activity concentration ( bq kg ) , respectively for their coefficients , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ g_{\text{t , r } } $ $ \end{document } is dose conversion coefficient for r radionuclide ( sv bq ) . dose conversion coefficients of ra , th , k and cs radionuclides for the adult members of society are 4.5 10 , 2.3 10 , 6.2 10 and 1.3 10 sv bq , respectively [ 13 , 15 , 16 ] . samples of fruits and vegetables produced and frequently consumed in the region were provided from a public market . any soil or foreign materials on the samples were removed so that they were suitable for consumption , divided into small pieces , and washed under distilled water . they were kept at room temperature for 3 months without allowing any contamination and then totally oven - dried at 105 c . the temperature of the oven was increased to 250 c and was continued until the samples were reduced to ash . the ashed samples were then homogenized and transferred into a plastic container ( 5 cm height 5 cm diameter ) . finally , the samples were sealed and stored for a period of about 1 month before counting , in order to allow equilibrium between ra and its short - lived decay products . the activity concentrations of ra , th , k and cs radionuclides in vegetable and fruit samples were determined using a gamma spectroscopic system , comprising a 2 2 nai(tl ) well - type detector and a detector surrounded by a cylindrical lead shield ( thickness , diameter and length approximately 3.5 , 13.7 and 15.5 cm , respectively ) . energy calibration of detector was performed by using co ( 37 kbq ) and ra ( 370 kbq ) point sources . ra , th , k and cs activity concentrations in vegetable and fruit were based on the detection of 609.3 , 583 , 1461 and 662 kev energy gamma rays transmitted by bi , tl , k and cs , respectively . ( 1)1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$a({\rm{bq}}\,{\rm{kg}}^ { { - 1 } } ) = \frac{c}{{m_{{\rm{s } } } \varepsilon p_{\gamma } } } $ $ \end{document}where c is the gamma ray count ( number per second ) , is the detector efficiency of the specific gamma ray , p is the absolute transition probability of gamma decay and ms is the mass of the sample ( kg ) . ingestion dose occurring through the intake of radionuclides depends on the consumption rate of foodstuff and the concentration of the radionuclide involved . ( 2 ) [ 3 , 13 , 14]2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ h_{\text{t , r } } = \mathop \sum \nolimits \left ( { u^{i } c_{\rm{r}}^{i } } \right)g_{\text{t , r } } $ $ \end{document}where i is foodstuff group , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ u^{\it 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}$$c_{\rm{r}}^{i } $ $ \end{document } are annual consumption rate ( kg ) and radionuclide activity concentration ( bq kg ) , respectively for their coefficients , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ g_{\text{t , r } } $ $ \end{document } is dose conversion coefficient for r radionuclide ( sv bq ) . dose conversion coefficients of ra , th , k and cs radionuclides for the adult members of society are 4.5 10 , 2.3 10 , 6.2 10 and 1.3 10 sv bq , respectively [ 13 , 15 , 16 ] . table 1 shows the natural and manmade radionuclide activity concentrations measured in samples of vegetables and fruits frequently consumed in elaz and its surrounding region . minimum detectable activity values for vegetable and fruit samples were calculated as 0.02 bq for th and cs ; 0.03 bq for ra ; and 0.1 bq for k. average activity concentrations of ra , th , k and cs of vegetable samples were 0.64 bq kg ( sd : 0.26 ) , 0.65 bq kg ( sd : 0.14 ) , 13.98 bq kg ( sd : 1.22 ) and 0.54 bq kg ( sd : 0.04 ) , respectively . the activity concentrations ranged between 0.11 and 0.99 bq kg for ra ; 0.470.84 bq kg for th ; 2.1444.77 bq kg for k ; and 0.170.79 bq kg for cs . average concentrations of ra for fruits were 1.52 bq kg ( sd : 0.34 ) and the values ranged between 0.73 and 2.81 bq kg . th concentrations ranged between 0.26 and 1.96 bq kg ( average 0.98 bq kg , sd : 0.23 ) . the average activities of k and cs radionuclides were 18.66 bq kg ( sd : 1.13 ) and 0.59 bq kg ( sd : 0.16 ) , respectively . k concentrations ranged between 1.34 and 35.49 bq kg.table 1activity concentrations of vegetables and fruitsidspeciesscientific nameactivity concentrations of vegetables and fruits ( bq kgfresh weight ) ra th k csvegetables f1bell pepper capsicum annuum l.bdlbdl7.21 0.910.48 0.04 f2parsley petroselinum crispum ( mill . ) hillbdlbdl44.77 1.90bdl f3scallion allium cepa l.bdl0.84 0.1729.41 1.85bdl f4pumpkin cucurbita moschata duchesne ex poir.bdlbdl2.14 1.36bdl f5leek allium ampeloprasum 0.64 0.37bdl10.02 1.15bdl f6radish raphanus sativus l.0.11 0.040.47 0.053.43 0.340.17 0.01 f7kale brassica oleracea acephalabdl0.64 0.245.78 1.57bdl f8capsicum capsicum annuum l.bdlbdl5.78 0.63bdl f9cabbage brassica oleracea capitata0.95 0.09bdl26.95 0.95bdl f10tomato solanum lycopersicum l.0.45 0.080.64 0.0910.73 0.70bdl f11eggplant solanum melongena l.0.99 0.19bdl16.57 1.600.79 0.06 f12lettuce lactuca sativa l.bdlbdl30.93 1.410.72 0.05 f13spinach spinacia oleracea l.0.80 0.33bdl9.84 0.92bdl f14peppermint mentha spicata l.0.60 0.36bdl2.22 1.05bdl f15garden cress lepidium sativum l.0.54 0.61bdl3.97 1.90bdlaverage0.64 0.260.65 0.1413.98 1.220.54 0.04fruits f16melon cucumis melo l.1.01 0.130.48 0.1335.49 0.990.53 0.04 f17pear pyrus spp.bdl1.96 0.3313.62 1.60bdl f18quince cydonia oblonga mill.2.81 0.451.14 0.3123.01 1.400.64 0.27 f19grapes vitis vinifera l.bdl0.26 0.121.34 0.63bdl f20watermelon citrullus lanatus ( thunb . ) matsum & nakaibdlbdl34.44 0.88bdl f21apple malus domestica borkh.0.73 0.451.04 0.264.04 1.25bdlaverage1.52 0.340.98 0.2318.66 1.130.59 0.16 bdl below detection limit activity concentrations of vegetables and fruits bdl below detection limit effective dose values exposed due to radionuclides taken into body through the consumption of fruit and vegetable samples are shown in table 2 . primarily , average activity concentration ( bq kg ) for each radionuclide was multiplied by food consumption rate , and annual activity intake value was determined in bq unit . food consumption rate was taken as 73 kg a for both fruits and vegetables . the effective dose value was then determined by multiplying annual activity intake value by effective dose coefficient . effective dose values of fruit samples for all radionuclides ( ra , th , k and cs ) were higher than those for vegetable samples . average effective exposure dose through the consumption of vegetable samples were 2.12 sv y ( sd : 0.86 ) , 11.04 sv y ( sd : 2.3 ) , 6.33 sv y ( sd : 0.55 ) and 0.51 sv y ( sd : 0.04 ) , respectively for ra , th , k and cs . effective dose values of ra , th , k and cs ranged between 0.36 and 3.25 , 7.89 and 14.10 , 0.97 and 20.26 and 0.16 and 0.75 sv y , respectively . average effective doses through the consumption of fruit samples were 4.99 sv y ( sd : 1.13 ) , 16.56 sv y ( sd : 3.91 ) , 8.44 sv y ( sd : 0.52 ) and 0.56 sv y ( sd : 0.16 ) , respectively for ra , th , k and cs . dose values ranged between 2.40 and 9.23 sv y for ra ; 4.37 and 32.91 sv y for th ; and 0.6116.06 sv y for k.table 2dose coefficients and committed effective dose values for ra , th , k and csradioisotopesactivity intake ( bq)effective dose coefficient ( sv bq)committed effective dose ( sv y)rangeaveragevegetables ra47 190.0450.36 0.133.25 0.622.12 0.86 th48 100.237.89 0.8414.10 2.8511.04 2.3 k1021 896.2 10 0.97 0.6220.26 0.866.33 0.55 cs39 31.3 10 0.16 0.010.75 0.060.51 0.04fruits ra111 250.0452.40 1.489.23 1.484.99 1.13 th72 170.234.37 2.0232.91 5.5416.56 3.91 k1362 836.2 10 0.61 0.2916.06 0.458.44 0.52 cs43 121.3 10 0.56 0.16 dose coefficients and committed effective dose values for ra , th , k and cs table 3 shows committed effective dose values reported for some countries and regions [ 3 , 1821 ] . total adult effective dose from vegetables and fruits frequently produced and consumed in elaz region for ra , th , k and cs radionuclides were calculated as 20 sv y ( sd:3.75 ) and 30.55 sv y ( sd:5.72 ) , respectively . in summary , this study found that adults living in the study region intake a radiation dose of approximately 50.55 sv y from fruit and vegetable consumption . this radiation dose ( 50.55 sv y ) is lower than the world average value ( 290 sv y ) and presents no risk to public health . dose values obtained in this present study reflect other reported values in general.table 3average effective dose values for elaz region and its comparison with literatureregion / countrycommitted effective dose ( sv y)referencesvegetablesfruitsfoodstuffnorth america110asia110europe110korean110jos plateau / nigeria(0.22,164)accra metropolitan area / ghana4,640rize / turkey22763elaz / turkey2030.55present study average effective dose values for elaz region and its comparison with literature ra , th , k and cs radionuclide concentrations in vegetables and fruits that are produced and frequently consumed in the elaz region of turkey were determined in this study . it was found that the radiation dose due to consumption of vegetables and fruits was less than the world average , and poses no threat to public health . the results were lower than the committed effective dose values reported for various regions and countries .

spontaneous medialstinal emphysema ( pneumomediastinum ) and pneumopericardium may be defined as the presence of free air or gas in the mediastinal structures and in the pericardial sac without an apparent precipitating cause . it most frequently occurs in young healthy adults without serious underlying pulmonary disease.although pneumomediastinum and pneumopericardium is often asymptomatic , it may cause pain in the neck and chest , dysphonia and shortness of breath . treatment is supportive unless the patient has a history of trauma from foreign body aspiration . the course of spontaneous pneumomediastinum and pneumopericardium is usually benign and self - limited.a case of spontaneous pneumomediastinum , pneumopericardium and subcutaneous emphysema in a 20-year - old male is reported in this paper .

spontaneous pneumomediastinum and pneumopericardium may be defined as the presence of free air or gas in the mediastinal structures and in the pericardial sac without an apparent precipitating cause . free air can enter mediastinal tissues by rupture of alveoli , laceration of the tracheobronchial tree or gastrointestinal tract , or passage of extraluminal gas into the thorax from the neck , retroperitoneum or chest wall . posteroanterior chest radiographs typically reveal a radiolucency between the left heart border and the mediastinal pleura . although pneumomediastinum is often asymptomatic , it may cause pain in the neck and chest , dysphonia and shortness of breath . although severe complications develop in some patients , the course of spontaneous pneumomediastinum is usually benign and self - limited . we report here a rare case of spontaneous pneumomediastinum , pneumopericardium and subcutaneous emphysema in a young adult , with a review of the literature . a 20-year - old male was admitted to the hospital with acute respiratory distress due to severe pain in the neck and chest during inspiration after having a spontaneous cough when he had been drinking a cup of coffee . he had no current history of trauma , aspiration of foreign body or drug abuse . the blood pressure was 150/100 mmhg , the pulse was 80 beats / min and respirations were 22/min . the crepitus by palpitation was remarkable along the left side of neck and anterior chest wall . the chest evaluation demonstrated symmetric , clear breath sounds and regular heart beats without murmur or hamman s sign . an arterial blood gas analysis in room - air was followed ; ph 7.432 , pao2 79.7 mmhg , paco2 45.9 mmhg , and sao2 96.4% . the hemoglobin was 12.5 g / dl , the hematocrit was 38% , the white cell count was 13,200/mm and the platelet was 231,000/mm . the chest and neck radiographs showed pneumomediastinum , pneumopericardium and subcutaneous emphysema in the neck and prevertebral space , but there was no evidence of pneumothorax ( figure 1 ) . the radiograph with gastrografin swallowing did not reveal any definite evidence of leakage from the esophagus and structural abnormalities ( figure 2 ) . the chest ct scan showed pneumomediastinum with extensive emphysema in the lower neck region with extension to the prevertebral soft tissue space and pneumopericardium . but there was neither a definite lung lesion nor any structural abnormalities in the bronchi and esophagus ( figure 3 ) . on the 7th hospital day , he had neither any symptoms of respiratory distress nor pain in the neck and chest . the follow - up radiographs and ct scan of the chest showed a complete resolution of pneumomediastinum , pneumopericardium and subcutaneous emphysema ( figure 4 ) . spontaneous pneumomediastinum is a rare and usually self - limited disease most commonly seen in young men and parturient women . an increased pressure gradient between the intra - alveolar and interstitial spaces enhances air leakage from small alveolar openings and ruptured alveoli into the perivascular adventitia yielding interstitial emphysema . however , because the visceral layers of the deep cervical fascia are contiguous with the mediastinum , air usually decompresses into the neck , preventing physiologically tamponade and pneumothorax . elevated pressure in the deep cervical fascia then favors dissection along blood vessels to other planes in the neck . dissection of the free air into the pericardial space is a common complication of barotrauma in neonates , but is very rare in adults where the apposition of pericardial layers is very tight . alveolar rupture occurs in the presence of elevated intra - alveolar pressure or damage to alveolar walls . causes of elevated alveolar pressure include airway obstruction ( e.g. , by mucous plugging in an asthmatic person or by a foreign body ) , mechanical ventilation ( particularly with a large ventilatory volume or high end - expiration pressure ) , blunt , trauma , coughing , emesis or valsalva maneuver ( e.g. , during parturition ) . causes of damage to alveolar walls include pneumonitis , emphysema , lung fibrosis and acute respiratory distress syndrome . causes of pneumomediastinum would be associated with the use of heroin marijuana , cocaine or nitrous oxide . other causes of pneumomediastinum include trauma , gas producing infections in the head , neck and abdomen , and surgery in the upper digestive tract as well as dental surgery . however , in a patient on a ventilator , it may occur by the same mechanism as that which causes most cases of pneumomediastinum , that is , by gas dissecting medially from interstitial emphysema of the lung . gas probably enters the pericardium along the venous sheaths , in which the collagenous support of the pericardial reflections is weak . they may experience dyspnea , dysphagia , neck and throat pain or dysphonia . physical examination may show respiratory distress , neck crepitus , decreased cardiac dullness , hamman s sign ( a mediastinal crunching sound that is synchronized with systole ) or , rarely , signs of pericardial tamponade . low grade fever is present in about one - third and mild leukocytosis in about one - half of cases . posteroanterior chest radiographs typically reveal a radiolucency between the left heart border and the mediastinal pleura . other findings may include highlighting of the aortic knob and the continuous diaphragm sign . pneumopericardium is usually manifested as a single band of gas that may outline both left ventricle and right atrium . if there is enough air , pneumopericardium can completely surround the heart and create the halo sign . esophagogram should also be performed to detect the possible esophageal tear if emesis or retching was the precipitating event . there are no pathognomic electrocardiographic changes in pneumomediastinum , although low voltage and axis shifts have been described . analgesics , bed rest and treatment for coughing are appropriate . breathing 100% oxygen will enhance reabsorption of the free air by increasing the gradient of nitrogen between the alveoli and the tissues . pain typically resolves within 1 to 2 days , and follow - up chest radiograph within 12 to 24 hours is necessary to detect any progression or complications such as pneumothorax . if progression occurs , the patient may require a needle aspiration or chest tube placement , but they are dangerous and provide only questionable benefit . in an adult with pneumopericardium , pericardial drainage should be performed only if there is hemodynamic embarrassment . in conclusion , spontaneous pneumomediastinum and pneumopericardum is a rare disease that occurs in a young healthy adult without underlying cause . absolute bed rest and a high concentration of oxygen supply were found to be a definite benefit .

objectives . allergic rhinitis is a common disease with increasing prevalence and high impact on economic burden and comorbidities . as treatment with pharmacological drugs is not always satisfactory due to side effects and incomplete efficacy , alternative treatment strategies are needed . ectoine is an osmolyte with membrane stabilizing and inflammation reducing capacities . nasal spray and eye drops containing ectoine are promising new treatment regimens for allergic rhinitis sufferers . design and methods . the current two noninterventional trials evaluated the efficacy and safety of ectoine containing nasal spray and eye drops for treating allergic rhinitis in comparison with either azelastine or cromoglycic acid containing products . nasal and ocular symptom developments as well as judgment of tolerability and efficacy were assessed both by investigators and patients over a time period of one to two weeks . results . both trials confirmed that ectoine containing products reduced nasal and ocular symptoms in allergic rhinitis patients . results clearly demonstrated good safety profiles of the ectoine products comparable to those of azelastine and even better to those of cromoglycate products . conclusion . ectoine containing nasal spray and eye drops are interesting new treatment strategies for sufferers of allergic rhinitis , combining both good efficacy and absence of side effects .

since it has great impact on patients ' quality of life , school performance , work productivity , and comorbid conditions such as asthma , it is considered as an important health problem . allergic rhinitis is defined as an allergic reaction ( most often ige - dependent ) to offending allergens such as dust mites , insects , animal dander , and pollens . symptoms include rhinorrhea , nasal obstruction , nasal and nasopharyngeal itching , sneezing , and postnasal drip . often , allergic rhinitis is accompanied by allergic conjunctivitis with ocular symptoms such as itchy and watery eyes , resulting in the term allergic rhinoconjunctivitis . according to its length of duration , allergic rhinitis is classified into intermittent ( symptoms present < 4 days a week of < 4 weeks ) and persistent ( symptoms present 4 days a week and for at least 4 weeks ) forms . symptom severity is used to classify allergic rhinitis into mild or moderate - severe forms . a number of pharmacological treatments of allergic rhinitis exist , such as , for example , oral and topical antihistamines , leukotriene receptor antagonists , intranasal glucocorticoids , and cromoglycic acid ( mast cell stabilizers ) . azelastine is a new - generation antihistamine applied topically as nasal spray or eye drops . it is used as treatment of allergic rhinitis , hay fever , and allergic conjunctivitis . although azelastine is regarded as effective possible first - line treatment for allergic rhinitis , common side effects , such as bitter taste of the drug and local irritation reactions and rare side effects such as fatigue or headache , can occur . cromoglycic acid is an antiallergic drug which inhibits the degranulation of mast cells , thereby blocking the release of inflammatory mediators . thus , cromoglycic acid prevents the development of allergic reactions rather than reducing acute symptoms and its onset of action is about four to seven days . due to its short half - life cromoglycic acid is thought to be a safe medication , and adverse events which might occur are usually mild , such as sneezing and sensation of burning . due to its good safety profile , cromoglycic acid can be prescribed for treating rhinitis in children and pregnant women . in general , many allergic rhinitis patients are still unsatisfied with the control of symptoms , complain about incomplete relief of symptoms , and suffer from unwanted side effects [ 5 , 6 ] . therefore , it is not surprising that increasing interest in the use of alternative and complementary medicine ( cam ) for treating rhinitis exists . thus , it was demonstrated that 40% of the american population uses cam , 17% of which uses it for treating otorhinolaryngologic diseases . however , so far no general recommendation for the use of cam can be given by aria guidelines as ambiguous study results are available . the present two individual studies compared treatment of allergic rhinitis with ectoine containing nasal spray and eye drops with azelastine containing products ( study 1 ) or treatment with ectoine containing nasal spray with that of cromoglycic acid containing nasal spray ( study 2 ) . ectoine is a natural amino acid derivate which is produced by bacteria living under extreme harsh environmental conditions where it serves as osmoregulatory compatible solute [ 9 , 10 ] . ectoine works via a mechanism called preferential exclusion [ 11 , 12 ] . if it is present together with proteins or lipids , ectoine is expelled from their surfaces , thereby increasing the hydration of the surface and stabilizing lipid layers . its membrane stabilizing as well as inflammation reducing capacities makes ectoine an interesting candidate for the treatment of allergic rhinitis . these studies served to investigate the efficacy and safety of ectoine containing nasal spray and eye drops in patients with allergic rhinitis . the current paper describes two noninterventional studies carried out with ectoine containing nasal spray and eye drops assessing their efficacy in comparison with azelastine nasal spray and eye drops ( study 1 , nct02131051 ) or cromoglycic acid nasal spray ( study 2 , nct02131038 ) . the ectoine eye drops contain an iso - osmotic solution with 2% ectoine and 0.35% hydroxyethyl cellulose ; the ectoine nasal spray is a hypertonic solution with 2% ectoine . additional ingredients of the eye drops were sodium chloride , sodium dihydrogen phosphate dihydrate , sodium monohydrogen phosphate dihydrate , and water . additional ingredients of the nasal spray were sodium chloride and water . in study 1 , both nasal spray and eye drops were used , whereas only the nasal spray was used in study 2 . the azelastine eye drops contain 0.5 mg / ml azelastine hydrochloride with one drop administering 0.015 mg azelastine hydrochloride , and the azelastine nasal spray contains 1 mg / ml azelastine hydrochloride with one puff administering 0.14 mg azelastine hydrochloride . additional ingredients of the eye drops were benzalkonium chloride ( preservative ) , sodium edetate , hypromellose , sorbitol , sodium hydroxide , and water . additional ingredients of the nasal spray were sodium edetate , hypromellose , citric acid , sodium chloride , sodium hydrogen phosphate , and water . during study 2 , the spray contained 20 mg / ml cromoglycic acid corresponding to 2.8 mg sodium cromoglycic acid per puff . in addition , the following ingredients were present in the formulation : benzalkonium chloride ( preservative 0.014 mg / puff ) , sodium edetate , sodium chloride , sodium dihydrogen phosphate , sodium monohydrogen phosphate , sorbitol , and water . on day 0 ( visit 1 ) patients were asked to participate in the study , and upon signing the informed consent form and patient information , they were allocated to one of the study groups , without any washout period . antiallergic medications used the last two days prior to inclusion were recorded by the physician . patients were treated either with ectoine containing nasal spray and eye drops or with azelastine containing nasal spray and eye drops . patients of the ectoine group had to apply one eye drop per eye and one puff of the nasal spray per nostril four times per day . patients of the azelastine group had to apply one eye drop per eye and one puff of the nasal spray per nostril twice per day . the treatment period was 7 days , and patients were asked to document their symptoms , together with possible comedication and adverse effects daily in patient diaries at the evening . therefore the patients ' assessments started after the products had been applied already . following treatment , patients came back for visit 2 ( day 7 ) , during which symptom scores were evaluated and tolerability , efficacy and compliance , and possibly comedications , antiallergic and others , were assessed . male or female patients aged 1870 with proven allergy and acute symptoms in nose and eye ( sum nasal score 15 and sum oral score 6 ) were allowed to take part in the study . exclusion criteria were pregnant and nursing women , drug addicts and persons unable to give consent to study participation , patients with intolerance against ingredients of any of the study treatments , previous eye or nose surgery , concomitant treatment with antiallergic drugs , and diseases which might influence the output of the study according to the physicians ' judgment . single nasal ( nasal obstruction , rhinorrhea , and sneezing ) and ocular symptoms ( eye itching , tearing , and conjunctivitis ) were scored with an 8 point scale ranging from no symptoms ( 0 ) to very severe symptoms ( 8) . efficacy , tolerability , and compliance were judged by using a scale ranging from 0 ( very good ) to 8 ( bad ) . thus , a general judgment , of either how well to tolerate or how efficient the products were , had to be given by the patients and documented in the patient diaries . both scoring values were based on the patients ' personal opinion / feeling with the products . whereas efficacy and tolerability were assessed both by patients and by physicians , further analysis was carried out with the mann - whitney u test , wilcoxon test , or friedman test . unavailable data were treated as missing values or substituted by the last value carried forward method . this study was designed as a crossover study , without any washout period within the first week . half of the patients received ectoine nasal spray whereas the other half received cromoglycic acid containing nasal spray . thus , patients who started with one week treatment with ectoine nasal spray received cromoglycic acid containing nasal spray within the second week and vice versa . for simplification reasons , patients starting their treatment with ectoine are termed group a , and the ectoine nasal spray had to be applied at least 5 times per day , whereas the cromoglycic acid spray had to be applied 4 times a day . thus , patients had to take the ectoine product at least 5 times a day but could upgrade dosing if they felt that medication was not sufficient . patients had to attend visits to the investigator on day 0 ( v1 ) , day 7 ( + 2 days ) ( v2 ) , and day 14 ( + 2 days ) ( v3 ) . during those visits , the investigator assessed nasal ( nasal obstruction , sneezing , and rhinorrhea ) and ocular symptoms ( eye itching , tearing , and conjunctivitis ) as well as palate itching and turbinate hyperplasia . at the end of the study ( v3 ) , efficacy , in addition to the investigator 's assessment , patients had to document daily their ocular and nasal symptoms as well as their judgment of tolerability and efficacy in a patient diary at the evening . based on the design the patients scoring started after the study medication had been applied . male or female patients with diagnosed allergy and moderate to severe acute symptoms of nasal obstruction , sneezing , and rhinorrhea were allowed to take part in the study . exclusion criteria were intolerance against ectoine or cromoglycic acid , pregnancy , previous nose surgeries , or ongoing treatment with additional antiallergic drugs . single nasal symptoms ( nasal obstruction , rhinorrhea , and sneezing ) and ocular symptoms ( eye itching , tearing , and conjunctivitis ) as well as the symptoms palate itching and turbinate hyperplasia were scored with an 8 point scale ranging from no symptoms ( 0 ) to very severe symptoms ( 8) . efficacy , tolerability , and compliance were judged by using a scale ranging from 0 ( very good ) to 8 ( bad ) . thus , a general broad judgment , of how well to tolerate and how efficient the products were , had to be given by the patients and to be documented in the patient diaries . both scoring values were based on the patients ' personal opinion / feeling with the products . whereas efficacy and tolerability were assessed both by patients and by physicians , compliance was solely judged by physicians . pollen score . in order to reflect the current pollen exposure , data from the online hexal pollen calendar were used to grade pollen exposure into mild , moderate , or severe ( 1 , 2 , or 3 ) scores during the course of the study . statistics . safety analyses were performed on the entire study population whereas efficacy analysis was performed on all patients who completed the treatment . further analysis was carried out with the mann - whitney u test , wilcoxon test , or friedman test . unavailable data were treated as missing values or substituted by the last value carried forward method . both studies were conducted in accordance with the declaration of helsinki . all investigations were carried out with the understanding and consent of all participants . this was a noninterventional trial taking place at two german ear nose throat ( ent ) practices starting in june 2010 and being completed in september 2010 . , 48 patients took part in the study , of which 43 were included in the final analysis ( 31 females and 12 males ) . mean age of patients was 35 years , and both groups were comparable in regard to clinical aspects . nasal symptom scores were assessed both as single symptoms and as sum of all nasal symptoms ( tnss ) . the mean symptom score of nasal obstruction decreased significantly by 45.95% in the ectoine group and by 49.23% in the azelastine group ( v1 to v2 , p < 0.001 for both groups ) . the documentation of the patient diaries also reflected a significant decrease by 18.39% in the ectoine group ( p = 0.003 ) and by 17.83% in the azelastine group ( p = 0.044 ) . mean values decreased by 56.88% in the ectoine group and by 52.50% in the azelastine group ( p < 0.001 for both groups ) . the patient documentation showed a clear decrease of the symptom rhinorrhea which , however , was not significant . values decreased by 28.75% in the ectoine group ( p = 0.054 ) and by 19.45% in the azelastine group ( p = 0.133 ) . the symptom sneezing decreased significantly from v1 to v2 : values decreased by 59.52% in the ectoine group and by 59.84% in the azelastine group ( p < 0.001 for both groups ) . the patient documentation also reflected the symptom decrease which was not significant in the ectoine group ( 25.61% , p = 0.475 ) but significant in the azelastine group ( 39.47% , p < 0.001 ) . nasal itching decreased significantly from v1 to v2 : values decreased by 76.40% in the ectoine group ( p = 0.001 ) and by 69.31% in the azelastine group ( p < 0.001 ) . according to the patient documentation , nasal itching scores decreased by 27.12% in the ectoine group ( p = 0.068 ) and by 42.38% ( p = 0.002 ) in the azelastine group . the sum of nasal symptom scores ( nasal obstruction , rhinorrhea , sneezing , and nasal itching ) showed a significant decrease from v1 to v2 ( as assessed by physicians ) : sum scores in the ectoine group decreased from 20.71 3.52 to 8.52 4.74 ( decrease of 58.85% ; p < 0.001 ) and sum scores in the azelastine group decreased from 21.73 3.34 to 9.32 6.24 ( decrease of 57.11% ; p < 0.001 ) . data are depicted in figure 2 . according to the patients ' assessment ( see figure 3 ) , values decreased by 23.05% in the ectoine group ( p = 0.076 ) and by 33.14% in the azelastine group ( p = 0.02 ) . ocular symptom scores were also assessed as single symptoms and as sum of all ocular symptoms ( toss ) . the symptom conjunctivitis clearly decreased from v1 to v2 , as reflected by decline of 48.15% in the ectoine group ( p = 0.058 ) and of 46.07% in the azelastine group ( p = 0.013 ) . in the patients documentation , scores of conjunctivitis decreased by 34.09% in the ectoine group ( p = 0.218 ) whereas an increase by 14.77% was observed in the azelastine group ( p = 0.885 ) . there was a significant decrease in the symptom scores of eye itching : in the ectoine group , the mean decreased by 48.15% ( p = 0.008 ) whereas values of the azelastine group decreased by 46.07% ( p = 0.002 ) . corresponding decreases as assessed by the patients were 17.65% in the ectoine group ( p = 0.604 ) and 5.33% in the azelastine group ( p = 0.14 ) . tearing . a statistical decrease in the scoring of the symptom tearing was also observed from v1 to v2 : in the ectoine group , values decreased by 52.46% ( p = 0.003 ) whereas values in the azelastine group decreased by 40.43% ( p = 0.039 ) . the patient documentation of the symptom tearing also showed a clear decrease of values ( 5.56% with p = 0.886 in the ectoine group and 18.63% with p = 0.357 in the azelastine group ) . the toss ( sum of conjunctivitis , eye itching , and tearing ) decreased significantly from v1 to v2 in both groups ( p < 0.001 for ectoine , p = 0.009 for azelastine ) . starting mean values at v1 were 9.43 3.14 in the ectoine group and 9.5 4.22 in the azelastine group which decreased by 45.96% to 5.10 4.38 in the ectoine group and by 44.98% to 5.23 4.36 in the azelastine group . decreases of toss values as assessed by patients were not significant ( figure 4 ) ( data not shown ) . palate itching . as for nasal and ocular symptoms , a clear decrease of the symptom palate itching was observed from v1 to v2 : in the ectoine group , values decreased from 2.52 2.71 to 1.19 1.72 ( p = 0.024 ) , and in the azelastine group , values decreased from 3.36 2.68 to 1.5 1.92 ( p = 0.018 ) . values of the patients ' documentation did only reach statistical significance in the azelastine group : here , the scoring decreased from 3.81 2.5 to 2.15 2.13 ( p < 0.001 ) . in the ectoine group , values decreased from 1.76 2.1 to 1.67 2.15 ( p = 0.854 ) . correlation of pollen count and nasal symptoms . in order to normalize the nasal symptoms ( nasal constriction , rhinorrhea , and sneezing ) to the pollen burden , a quotient from sum score and pollen counts was determined . values of quotients decreased significantly from 8.97 3.98 to 5.23 3.59 in the ectoine group ( p = 0.002 ) and from 9.73 3.59 to 5.76 5.26 in the azelastine group ( p = 0.011 ) , thus confirming the decrease of nasal symptoms during the pollen season upon treatment . the physicians ' assessment of efficacy of both products was similar at v2 , and with values of 2.48 ( good ) in the ectoine group and 2.64 ( good - satisfactory ) in the azelastine group , there was no significant difference between groups . the general tolerability was assessed as very good to good in both groups ( 1.33 in the ectoine group and 1.45 in the azelastine group ) , and the compliance was comparably good ( values < 1 ) in both groups . values of the patients ' assessments of efficacy and tolerability are shown in figures 5 and 6 . the patients ' evaluations resulted in comparable values of efficacy and tolerability without statistical differences between treatment groups . comparison of reduction of symptoms between groups . in order to calculate if reduction of symptoms from v1 to v2 was different between the treatment groups , differences of mean v1 and v2 values were compared via mann - whitney u test . as shown in table 3 , there were no statistical differences between the ectoine and the azelastine group , thus confirming that both substances worked comparably well . the same calculation was performed for the patient data . here , no statistical difference was shown except for the symptom palate itching . adverse events ( aes ) . in total , 8 aes occurred during the study ( see table 5 ) . 2 aes occurred in the ectoine group , whereas 6 aes occurred in the azelastine group . this was a noninterventional trial taking place at a german ear nose throat ( ent ) practice starting in may 2009 and being completed in september 2009 . , 50 patients ( 33 females and 17 males ) with an average age of 34 years took part in the study . both treatment groups were homogeneous from a clinical point of view . nasal obstruction . both patient groups started with a comparable mean nasal obstruction score of 5.80 in group a and 5.64 in group b ( physician 's assessment ) . the symptom scores decreased to 3.2 ( group a ) and 3.44 ( group b ) after a week , and a further decrease to 2.52 ( group a ) and 2.92 ( group b ) was observed after 2 weeks . decreases were significant in both groups with p values both for 1 week and for 2 weeks of p < 0.001 . similarly , patient scores of the symptom nasal obstruction decreased from 4.08 ( group a ) and 3.60 ( group b ) on day 0 to 2.84 ( group a , p = 0.009 ) and 3.24 ( group b , p = 0.464 ) on day 7 and further to 2.52 ( group a , p = 0.004 ) and 2.56 ( group b , p = 0.041 ) on day 14 . the symptom rhinorrhea decreased significantly ( p < 0.001 ) for both groups both from v1 to v2 and from v1 to v3 according to the physician 's assessment . values decreased from 5.12 to 2.40 ( v2 ) and further to 1.88 ( v3 ) in group a and from 4.96 to 2.68 ( v2 ) and to 2.76 ( v3 ) in group b. according to the patients ' evaluation , scoring of rhinorrhea decreased from 3.12 to 2.32 ( d7 , p = 0.104 ) and further to 2.04 ( d14 , p = 0.010 ) in group a. in group b , values decrease from 3.80 to 3.08 ( d7 , p = 0.115 ) and further to 2.28 ( d14 , p < 0.001 ) . the symptom sneezing also decreased significantly ( p < 0.001 ) from v1 to v2 and from v1 to v3 in both groups . baseline scores from group a were 5.72 and decreased to 2.56 ( v2 ) and further to 1.80 ( v3 ) , whereas values in group b decreased from 5.68 to 2.80 ( v2 ) and to 2.6 ( v3 ) . according to the patients ' evaluation , scoring of the symptom sneezing decreased from 3.16 to 2.44 ( p = 0.20 ) on day 7 to 2.12 ( p = 0.265 ) on day 14 in group a , whereas values decreased from 4.04 to 2.64 ( p = 0.018 ) on day 7 to 2.40 ( p < 0.001 ) on day 14 in group b. to reflect the development of the sum of nasal symptoms , the total nasal score ( nasal obstruction , rhinorrhea , and sneezing ) was calculated . results are depicted in figures 8 and 9 . according to the physician 's assessment , tnss scores decreased significantly for both groups both from v1 to v2 ( p < 0.001 ) and from v1 to v3 ( p < 0.001 ) . scores assessed by patients showed that decreases in tnss from d1 to d7 were not significant whereas significant decreases in tnss scores from d1 to d14 were shown both for group a ( p < 0.001 ) and group b ( p < 0.001 ) . to investigate the development of ocular symptoms during the treatment period , the single symptoms eye itching , tearing , and conjunctivitis / redness of eyes were assessed both by the investigator and by the patients . details of scores are listed in tables 6 and 7 . according to the investigator 's assessment , all observed ocular symptoms improved significantly from v1 to v3 in group a , whereas only the symptoms eye itching and tearing improved significantly in group b. the patients ' assessment of ocular symptoms showed that the symptoms eye itching and eye redness improved significantly in group a , whereas decreases in symptom scores from day 1 to day 14 were not significant in group b. the development of the sum of ocular symptoms ( eye itching , conjunctivitis , and tearing ) as assessed by the investigator is depicted in figure 10 . it could be confirmed that ocular symptoms decreased significantly from v1 to v2 ( p < 0.001 for group 1 ; p = 0.008 for group b ) as well as from v1 to v3 ( p < 0.001 for group 1 ; p = 0.003 for group b ) . the development of total ocular symptom score as assessed by patients is shown in figure 11 . here , a significant decrease of symptom score was only observed in group a from day 1 to day 14 ( p = 0.026 ) . in addition to nasal and ocular symptoms , the development of the symptom palate itching was determined both by the investigator and the patients . as shown in table 8 , significant decreases in the symptom palate itching in contrast , patients ' assessment of this symptom showed only small decreases in this symptom which were not significant . additionally , the development of turbinate hyperplasia was determined by the investigator . as shown in table 8 , treatment resulted in a significant improvement of this symptom within the first week of treatment which was still significantly improved after two weeks of treatment . no differences between groups could be determined for this symptom ( table 9 ) . correlation of pollen count and nasal symptoms . in order to rule out that results might be influenced by the existence of pollens , the quotient of tnss values and pollen count scores confirmed a significant decrease of tnss values both from v1 to v2 ( p < 0.001 ) and from v1 to v3 ( p < 0.001 ) ( data not shown ) . good to satisfactory with a score of 2.68 1.89 ( group a ) and 2.96 1.72 ( group b ) at v2 and a score of 3.12 2.11 ( group a ) and 2.80 2.06 ( group b ) at v3 . similarly , the patients ' assessment of efficacy was good to satisfactory both on day 7 ( group a : 2.76 1.89 ; group b : 2.96 1.81 ) and on day 14 ( group a : 2.56 2.00 ; group b : 2.44 2.16 ) without statistical differences between groups . following 1 week of treatment , the tolerability was judged as very good in group a ( 1.24 1.30 ) and as good ( 2.40 1.53 ) in group b. following crossover of groups , tolerability of the treatment was judged as satisfactory ( 3.0 2.16 ) in group a and as very good ( 0.88 1.05 ) in group b. the changes of tolerability between both groups were highly significant ( p < 0.001 ) , thus indicating that tolerability was significantly better following a 7-day treatment with ectoine containing nasal spray in comparison to 7-day treatment with cromoglycic acid nasal spray . those differences in tolerability scoring were also clearly visible in the patients ' assessment of tolerability . whereas tolerability was judged as very good ( 1.30 1.48 ) within the first days of treatment in group a , scoring for the second week decreased to a mean score of 2.65 1.89 corresponding to good to satisfactory . group b showed the opposite development with a tolerability scoring of 2.35 1.68 ( good ) within the first 7 days which improved to a mean score of 0.97 1.24 ( very good ) within the second half of the treatment . details of the patients ' tolerability assessment are depicted in figure 12 . in summary , patients judged the tolerability significantly better under treatment with ectoine containing nasal spray compared to treatment with cromoglycic acid nasal spray ( p < 0.001 ) . the compliance was assessed as very good by the physician , and values were not statistically different between groups ( see table 10 ) . in contrast , 13 patients complained about a burning sensation during treatment with cromoglycic acid nasal spray . the correlation between the observed aes ( burning sensation , displeasing smell , and dehydration effect ) was judged as probable . the current studies investigated the efficacy and safety of ectoine containing nasal spray and eye drops in comparison with commonly applied pharmacological treatments of allergic rhinitis . in two noninterventional trials , although this paper covers results of two separate studies , they were summarized in one document as the indication was very similar and both studies aimed to compare ectoine containing products with other topical medications . as the study with cromoglycic acid was one of the first studies with ectoine products , dosage was slightly higher than in the study comparing ectoine and azelastine products . as a placebo - controlled , randomized trial with ectoine nasal spray and eye drops which was conducted after the study 2 had confirmed that the dose of 4 uses per day was sufficient to show significant superiority over placebo treatment , this dosage was chosen in study 1 . both studies demonstrated that allergic rhinitis can be successfully and safely treated with ectoine containing products , thus offering a potential new treatment strategy for allergic rhinitis sufferers . although this study design forbids randomization of patients , use of placebo , and blinding of study medication , it still reflects the most realistic standard clinical practice . thus , patients were included independently on their prior medication and no washout period had to be kept . in order to ensure homogeneity of patients , all had to show a certain degree of symptoms at inclusion reflected by a minimum of tnss values . additionally , symptom scores were correlated with pollen count scores in order to include objective measures into the analysis . importantly , sites specialized in the area of ear nose throat practice were chosen to warrant a very precise assessment of symptoms by specialized physicians and to have a homogeneous patient population . although we believe that valuable results can be drawn from noninterventional trials , one drawback of this study design is the fact that one can not include a placebo group into the study population . on the other hand , it has been demonstrated that double - blind randomized placebo - controlled trials clearly have their limitations and disadvantages and that particularly patients ' awareness of a placebo arm can lead to modifications of results due to patients ' expectations and interpretations . this was confirmed by a comparison of open and controlled study designs in neuroleptic studies , indicating that results of well performed open studies earn more attention . in study 1 , it was shown that both the ectoine and the azelastine products resulted in a clear decrease of symptoms of allergic rhinitis over the study period of 7 days . according to the physicians ' evaluation , the symptoms nasal obstruction , rhinorrhea , sneezing , nose itching , conjunctivitis ( azelastine group only ) , the mean decrease of tnss was 58.85% in the ectoine group and 57.11% in the azelastine group , thus demonstrating a strong clinical relevance . similarly , mean decreases in toss were 45.96% in the ectoine group and 44.98% in the azelastine group and therewith reflect strong clinical relevance , too . study 2 also demonstrated a significant decrease of symptom scores upon treatment : within the first week of the study , tnss values decreased by 50.96% ( group a ) and by 45.21% ( group b ) , and decreases within the entire study period of 2 weeks were 62.74% ( group a ) and 49.14% ( group b ) according to the physician 's assessment . nasal obstruction is often caused by an enlargement of the nasal turbinates which are located on the lateral walls on each side of the nose . thus , the significant improvement of turbinate hyperplasia as assessed by the physician underlined the efficacy of both treatments in reducing nasal obstruction . in comparison to the physicians ' assessment of symptoms , this might be most likely due to the fact that starting symptom values as assessed by patients themselves were lower than the physicians ' values . this in turn is at least partly accredited to the fact that patients ' first assessments of symptoms were documented at the end of the first treatment days whereas physicians documented baseline symptoms during the first site visit prior to the start of treatment . a recent placebo - controlled study in an environmental challenge chamber showed that 3 hours after application of the ectoine nasal spray and eye drops the symptoms were decreased by ~20% . this decrease reflects roughly the difference between the first assessment by the physicians and the first patient diary entry . in addition , physicians are able to carry out ranking of symptoms based on their experience with many patients ; thus , their judgment might be considered more objectively . on the other hand , symptoms such as itching of eyes , nose , and palate can not be measured with a scientifically valid method and are thus prone to personal perception and difficult to be assessed by physicians together with patients . taken together , an overestimation by the physician or an underestimation by the patients is not likely . in study 1 , the patients ' assessment showed that for the azelastine group , decreases were significant for the symptoms nasal obstruction , sneezing , and nasal itching . for the ectoine group , values decreased significantly in the symptom nasal obstruction , whereas a clear but not significant decrease in the symptoms nasal itching , sneezing , and rhinorrhea was observed . in total , decreases of tnss were 24.68% in the ectoine group and 35.26% in the azelastine group , thus confirming a clinical relevance of the treatment . clear decreases in the ocular symptoms tearing and eye itching were assessed by patients of both groups ; however , values did not reach significance . the symptom conjunctivitis was clearly ( but not significantly ) decreased in the ectoine group , whereas it became slightly worse in the azelastine group . in total , toss as assessed by patients decreased by 19.57% in the ectoine group and by 11.81% in the azelastine group . although no ocular treatment was applied in study 2 , ocular symptoms of allergic rhinitis clearly improved upon treatment with ectoine or cromoglycic acid nasal spray . according to the physicians ' assessment , toss values decreased by 59.09% in group a and by 39.32% in group b after 1 week of treatment and by 73.74% in group a and by 45.47% in group b after 2 weeks of treatment . it is not surprising that local and nonpharmaceutical nasal treatment might also influence ocular symptoms as recent studies suggest a crosstalk between the nose and eyes . the mechanism of the influence of symptoms via the nasolacrimal duct is not fully understood but thought to be via a mucosal connection , possibly via a nose - eye reflex . the patients ' assessment of ocular symptom development in study 2 confirmed a significant decrease in the symptom eye itching after two weeks ' treatment in both groups . a significant reduction of the symptom eye redness was observed in group a but not in group b. however , as ocular symptom scores were generally rather small in this study and treatment aimed mainly to reduce nasal symptoms , further studies will be needed to evaluate the efficacy of treatments on ocular symptoms . interestingly , another published study with azelastine eye drops , cromoglycate eye drops , or placebo eye drops showed superiority of both active treatments versus placebo without significant differences between the two active treatments , and a future study comparing treatment with ectoine eye drops only with other pharmacological eye drop formulations would be desirable . the current studies both showed that ectoine nasal spray and eye drops can safely be applied in patients with allergic rhinitis . patients judged the tolerability of the products as similarly good as the azelastine products and significantly better than cromoglycic acid nasal spray , and the very low numbers of aes reflected a very good safety profile of the used treatments . the crossover design of study 2 bears difficulties as no washout period between the crossover was carried out . however , as symptoms were assessed on a daily basis , effects following one treatment only ( after one week ) can be analyzed separately from the results following two treatments . as clear improvements of symptoms were already observed after one week of treatment , this time span seems sufficient to evaluate effects of either treatment a or b. as the efficacy and safety of azelastine has been studied in a huge range of clinical trials during the last decades , one can use historical data to bring the results of the current study into a broader context ( for results from comparator studies see table 11 ) . in addition , results from placebo groups of comparator studies can be used in order to rank the current results . thus , comparable data confirmed a superiority of azelastine versus placebo treatment , and values indicate that effects of azelastine are usually about 2 - 3-fold higher than placebo . however , comparing the actual values of the current study with other studies is rather difficult as design ( e.g. , randomized versus nonrandomized , placebo - controlled versus not placebo - controlled , differences in length of treatment , and differences in scaling of symptoms ) and dose ( azelastine concentration and number of daily applications ) of available studies differs enormously . in addition , most studies used nasal spray only and assessed solely nasal symptoms , whereas the current study is one of few studies acknowledging also ocular symptoms of allergic rhinitis . taken together , results of the current study 1 showed that effects of ectoine containing products are almost comparable with those of azelastine , a well - studied drug , which has a proven superiority to placebo treatment and is commonly prescribed against allergic rhinitis . cromoglycic acid has been a common drug in treating allergic rhinitis , and although it is thought not to be as effective as intranasal steroids or antihistamines , it has been shown to reduce both nasal and ocular symptoms and it is therefore a reasonable therapy option . in particular , its good safety profile makes it an interesting treatment option both for children and pregnant women . as no published studies were identical with the current study design ( study 2 ) in terms of treatment duration and analysis of end points , only general comparisons to cromoglycic acid studies can be drawn . several studies have confirmed that cromoglycic acid is superior to placebo in patients with allergic rhinitis . thus , schuller and colleagues investigated the efficacy of cromolyn sodium in comparison to nedocromil sodium and placebo . over a treatment period of 8 weeks , it was demonstrated that cromolyn resulted in a clear improvement of nasal symptoms , particularly in the symptom stuffy nose . a further placebo - controlled study confirmed that cromoglycate acid nasal spray provided significant relief of nasal symptoms within 2 weeks of treatments which were significant for the symptoms sneezing and nasal congestion and clearly visible but not significant for the symptoms rhinorrhea and nasal pruritus . taken together , ectoine containing nasal spray and eye drops have been demonstrated to be promising alternatives to pharmacological drugs with both good efficacy and a very good safety profile . as the ectoine nasal spray and eye drops act purely physically on the nasal and ocular mucosa , it makes those products particularly interesting for patients with reservations about pharmacological therapy . an additional study in children and adolescents with seasonal allergic rhinitis ( data not yet published ) has confirmed the safety of ectoine containing nasal spray and eye drops in the pediatric population . further studies should be undertaken to further investigate the onset of action and compare it to commonly applied pharmacological drugs . quick relief of symptoms is crucial for patients and understood to be advantageous when comparing , for example , azelastine with intranasal corticosteroids and should therefore be assessed for the ectoine products . a controlled , ramdomised study which was carried out using a controlled environmental exposure chamber showed a quick onset of action of ectoine nasal spray and eye drops and confirmed the efficacy in reducing both nasal and ocular symptoms . additional studies applying the ectoine eye drops only are needed to further elucidate their impact on ocular symptoms during allergic rhinitis .

for a little over a decade , omics methods ( transcriptomics , proteomics , metabolomics , and physionomics ) have been used to discover and probe the mode of action of both synthetic and natural phytotoxins . for mode of action discovery , the strategy for each of these approaches is to generate an omics profile for phytotoxins with known molecular targets and to compare this library of responses to the responses of compounds with unknown modes of action . using more than one omics approach enhances the probability of success . generally , compounds with the same mode of action generate similar responses with a particular omics method . stress and detoxification responses to phytotoxins can be much clearer than effects directly related to the target site . clues to new modes of action must be validated with in vitro enzyme effects or genetic approaches . thus far , the only new phytotoxin target site discovered with omics approaches ( metabolomics and physionomics ) is that of cinmethylin and structurally related 5-benzyloxymethyl-1,2-isoxazolines . these omics approaches pointed to tyrosine amino - transferase as the target , which was verified by enzyme assays and genetic methods . in addition to being a useful tool of mode of action discovery , omics methods provide detailed information on genetic and biochemical impacts of phytotoxins . such information can be useful in understanding the full impact of natural phytotoxins in both agricultural and natural ecosystems .

understanding the modes of action of natural compounds as toxicants is important for at least two reasons . from an ecological and evolutionary standpoint , knowing the mode of action of such compounds is critical to understanding the function of the compound in nature . for example , the high activity of phytotoxins from plant pathogens on specific molecular targets found in green plants , but not in fungi , provides strong evidence that the compounds have evolved as virulence factors for the pathogens ( duke and dayan , 2011 ) . from a more practical standpoint , natural compounds often have been the source of pesticides with new modes of action ( dayan et al . , 2012 ) , and new modes of action are needed to combat the increasingly rapid evolution of pest resistance to the currently used pesticides , particularly herbicides ( duke , 2012 ) . there are only about 20 molecular targets for the herbicides currently used ( duke , 2012 ) , but natural phytotoxins have many effective modes of action that are not used by commercial herbicides ( duke and dayan , 2011 ) . exploitation of a natural product mode of action may or may not involve the use of the natural products as a chemical scaffold for the design of synthetic compounds that have the same mode of action . for example , the allelochemical leptospermone was the chemical basis for the design of the structurally similar triketone herbicides that inhibit hydroxyphenylpyruvate dioxygenase ( hppd ) ( beaudegnies et al . , 2009 ) , the last commercial herbicide mode of action introduced ( duke , 2012 ) . however , the phytochemical ryanodine only gave the clue for the new insecticide mode of action ( targeting ligand - gated calcium channels of insect muscles ) , for which structurally unrelated insecticides have been developed and commercialized ( lahm et al . , 2009 ) . the modes of action of most natural phytotoxins are unknown , especially for compounds thought to be involved in plant / plant interactions ( allelochemicals ) . there are many papers purporting to show the mode of action of herbicides and natural phytotoxins that have turned out to be wrong . there is no detailed and sure procedure for determination of the mode of action of a phytotoxin , although there are papers that will give researchers a start by using simple physiological assays ( e.g. , dayan et al . , 2000 ; dayan and watson , 2011 ; dayan and zaccaro , 2012 ) . omics methods ( genomics paired with transcriptomics and proteomics , as well as metabolomics , and physionomics ) offer new approaches to narrowing the search for the molecular target of a toxicant . robust technology for the first three of these approaches has only become available during the past decade , as evidenced by the fact that virtually all of the papers cited in this review have been published during the past 10 years . similar strategies generally are used in the utilization of all of these approaches for mode of action research . genomics underpins transcriptomics and proteomics , but genes are fixed , and treatment with a toxin does not change this . however , reverse genetics , that is determination of gene function by analysis of phenotypic effects of gene expression , is important in annotation of sequenced genes . affecting plant gene expression with phytotoxins can be valuable in this endeavor . the simplest method to use omics methods to probe the mode of action of a phytotoxin is to simply examine the effect of the compound on profiles of mrna , proteins , metabolites , or physiological processes at different doses and at different times after administering the dose and look for a hint about the molecular target . this approach has not been very productive , as the complexities of the responses to a toxicant at any level seldom reveal its initial target . even when the mode of action is known , this approach does not always point unambiguously to that target . the other and more rigorous approach is to generate a complete database of responses to a library of toxicants with known molecular targets . then , the detailed response to a compound with an unknown target can be compared with responses of plants to compounds with known targets . if the omics data do not fit known modes of action , they may suggest a new molecular target . few labs other than industrial ones have the capacity to generate and analyze large databases for all of the known modes of action . in this short review , we discuss the most commonly used omics approaches for the study of mode of action , giving the advantages and limitations of each . profiling of a global transcriptional response to a toxicant can be determined readily with the microarray or next generation sequencing technologies that have become available relatively recently . transcription profiling is used extensively in combinations with other methods to determine the mode of action of pharmaceuticals ( e.g. , bharucha and kumar , 2007 ; gobert and jones , 2008 ; pan et al . , 2008 ) . inhibition of an enzyme involved in a critical biochemical pathway or other protein receptor ( e.g. , the d-1 protein of photosystem ii ( psii ) ) by a chemical inhibitor should result in compensation through alteration of the transcription of genes encoding enzymes of the directly affected pathway , as well as genes encoding proteins associated with stress and with inactivation of xenobiotic compounds by chemical alterations . genomic information , coupled with microarray technology or next generation sequencing , allows us to readily examine the relative effect of an inhibitor on every gene in a genome . to get maximal information the first plant species for which this information was available was arabidopsis thaliana , and , accordingly , most of the first papers on global transcriptional responses to phytotoxins used this species for such studies ( e.g. , lechelt - kunze et al . , 2003 ; manfield et al . , 2004 ; baerson et al . , 2007 ; das et al . , 2010 , ) . the relatively small genome of a. thaliana reduces the amount and complexity of data that must be analyzed . the genomes of other plant species are becoming available at an increasing rate , and the annotations of those genomes are becoming more robust . some have argued that even though a. thaliana has not been a good plant species for discovery of new herbicide target sites by knocking out genes , it should be good for elucidating the mode of action of new phytotoxins to which it is sensitive by using functional genomics and metabolomics ( gressel , 2009 ) . at this time , the small genome , superior gene annotation , and rapidity with which experiments can be done with a. thaliana make it the best model organism for mode of action transcriptome fingerprinting . the transcriptome response is differentially affected by different doses of the phytotoxin , and the effects change over time . so , some uniform method of treatment must be used when generating a transcriptome response library to phytotoxins with different modes of action , such as treatment with the i50 and/or i80 ( concentrations required for 50 or 80 % inhibition of growth ) dose of the toxicant , with sampling at various times after the beginning of exposure . still , different compounds act at different rates due to several factors , including target sites , metabolic inactivation rates , uptake rates , and translocation rates . to complicate things even more , some phytotoxins act primarily on meristems ( e.g. , mitotic inhibitors ) and others act almost exclusively on green , photosynthesizing tissues ( e.g. , ps ii inhibitors ) ( fedtke and duke , 2005 ) . so , there is no perfect way to have a uniform method of building a transcriptome response library for phytotoxins with known molecular target sites . a good example of the difficulties is that of the work on the transcriptional responses of a. thaliana to the allelochemical benzoxazolin-2-(3h)-one ( boa ) ( baerson et al . , 2005 ) . the original intent of this work was to provide indications of the mode of action of this compound . from a dose response experiment , the i50 and i80 concentrations of boa on root growth of 10-day - old a. thaliana seedlings were determined , and the transcriptome responses of the seedlings were determined by whole transcribed genome microarrays at these two doses 24 hr after treatment . at this time and at these doses , almost 200 genes representing 12 functional categories genes involved with metabolism and cell rescue and defense accounted for more than half of the affected genes . many of these genes encode detoxification enzymes that also were induced by a set of structurally diverse xenobiotic compounds with different modes of action . non - phytotoxic xenobiotics that protect plants from herbicides ( safeners ) have similar effects on transcription of genes involved in phytotoxin detoxification in a. thaliana ( baerson et al . ( 2005 ) study , there was no clear indication of association of any of the genes affected by boa with a particular mode of action . separating effects of genes closely associated with the target site from those resulting from metabolic perturbation and defense pathways is a daunting task . genes affected at time points that will show earlier effects before the cascade of non - specific responses are more likely to be enriched in those more directly associated with a primary response . early work examined the effects of herbicides on a limited number of genes such as the studies of glombitza et al . ( 2004 ) , who examined the effects of two herbicide classes on 267 a. thaliana genes , and pasquer et al . ( 2006 ) , who studied the effect of herbicides from three chemical classes on wheat gene expression using a microarray with 600 barley cdnas . to truly determine the action of a phytotoxin on the transcriptome , a microarray composed of most or all ( global ) of the genome of the plant being studied must be used . there are a number of papers on transcriptional responses to herbicides with known modes of action . the earliest paper to use a global gene chip to probe the mode of action of a herbicide was that of lechelt - kunze et al . . a gene of a. thaliana encoding a putative fatty acyl - coa reductase involved in long - chain fatty acid alcohol biosynthesis was up - regulated by two herbicides ( flufenacet and benfuresate ) that inhibit very - long - chain fatty acid elongases as their mode of action . ( 2004 ) found several genes of a. thaliana that are closely associated with cell wall assembly to be involved in the mode of action of the cellulose synthesis - inhibiting herbicide isoxaben . zhu et al . ( 2008 ) determined transcriptome responses of glyphosate - resistant ( r ) and -susceptible ( s ) soybean near isogenic varieties . the resistance was imparted by a transgene encoding a glyphosate - resistant target site enzyme ( 5-enolpyruvylshikimate-3-phosphate synthase , epsps ) . at 1 , 4 , and 24 hr after treatment , 3 , 170 , and 311 genes were affected at these times , respectively , in the s variety , and 1 , 4 , and 24 genes were affected , respectively , in the r variety . the genes affected in the s variety would not have pointed clearly at epsps as the target site of glyphosate . there was no effect on transcription of epsps at any time point , and the only gene of the shikimate pathway affected was that of 3-deoxy - d - arabino - heptulosonate-7-phosphate synthesis , the first enzyme of the pathway . this effect was seen only at 4 hr . three other genes associated with aromatic amino acid metabolism were slightly affected , but no more so than many other unrelated genes . the results of this paper would probably not have been useful in discovery of the target of glyphosate if it were not already known . however , it did support the view that glyphosate has no other target site than epsps , in that there were no major transcriptome effects of glyphosate in the r variety . in a similar paper , 2007 ) , using imidazolinone herbicide- ( inhibitors of acetolactate synthase , als ) susceptible and resistant ( via a mutation in csr1 - 2 that encodes a subunit of als ) a. thaliana , found many genes affected by the herbicide in the s plants , whereas there were no significant changes to the transcriptome of treated r plants . nevertheless , the results would not have pointed the branched chain amino acid pathway as the site of the target of this type of herbicide . ( 2010 ) reported that transcriptional responses to glyphosate compared to those elicited by a series of als inhibitor herbicides in a. thaliana and brassica napus were quite different , so that one could differentiate between the two modes of action , as well as from other stress treatments . three classes of als inhibitors were used ( two sulfonylureas , one imidazolinone , and one triazolopyrimidine ) , and results suggested that the method could differentiate the between effects of compounds with the same molecular target , even when the compounds are from the same chemical family . they also reported that herbicidal inhibitors of ps i , ps ii , phytoene desaturase , auxin transport , and gibberellin biosynthesis have distinctive transcriptional signatures . transcriptional responses to other phytotoxins with known modes of action include the work of zhu et al . ( 2007 ) on the two psii - inhibiting herbicides atrazine and bentazon , and of raghavan et al . kelley et al . ( 2006 ) found a strong transcriptional response of gh3 , a primary auxin - responsive gene , in soybeans to the auxinic herbicides dicamba and clopyralid . the gene was not significantly affected by heat , drought , and salt stress , nor by virus infection . jamers and de coen ( 2010 ) found strong transcriptional effects on chlamydomonas reinhardtii genes associated with oxidative stress when the alga was treated with subtoxic levels of paraquat , a herbicide that causes all of its effect through rapid generation of reactive oxygen species . there are very few papers on the transcriptional responses of plants to allelochemicals or other phytochemicals . ( 2005 ) in which the main transcriptome responses were upregulation of stress genes and those associated with metabolic detoxification of xenobiotics . 2011 ) attempted to learn more of the mode of action of juglone ( 5-hydrox-1,4-napthoquinone ) by transcriptome profiling . the major effects were on genes related to cell growth , cell wall formation , detoxification of xenobiotics , abiotic stress , and epigenesis . the mechanism of action of l - dopa was probed by microarray ( golisz et al . , 2011 ) . the authors concluded that it has two mechanisms : 1 ) disruption of amino acid metabolism and 2 ) regulation of metal ion homeostasis , especially that of iron . ( 2008 ) probed the transcriptome response of a. thaliana to fagomine , gallic acid , and rutin , all putative allelochemicals of buckwheat ( fagopyrum esculentum ) . most of the responses were consistent with stress effects , but the target sites of the compounds were not identified . the natural phytotoxin cantharidin strongly affected transcription of the genome of a. thaliana , but this compound inhibits all 19 serine / threonine protein phosphatases of the plant ( bajsa et al . , 2011a , b ) . these enzymes strongly influence many signaling pathways , and , thus , 10 % of the genes of the a. thaliana genome were significantly affected within 24 hr of exposure to a cantharidin dose that reduced chlorophyll levels by 30 % . a large number of genes were both down- and up - regulated , and the pattern of genes affected changed dramatically with time after treatment as seen by a hierarchical clustering of microarray data ( fig . 1 ) . similar results were found with endothall , a commercial herbicide that is a close chemical analogue of cantharidin , which also inhibits serine / threonine protein phosphatases ( bajsa et al . the natural phytotoxin coronatine , an analog of the hormone methyl jasmonic acid ( meja ) , influenced 35 % of the genes regulated by meja in tomato ( uppalapati et al . , just as found for cantharidin , analysis of transcriptome responses to coronatine revealed major effects on signaling via the jasmonic acid , ethylene , and auxin pathways . coronatine and meja were concluded to share similar but not identical activities that impact multiple hormone pathways.fig . 1hierarchical clustering of microarray data ( heat map ) from the analysis of arabidopsis thaliana genes which changed expression 2 , 10 , 24 hr after treatment with 200 m cantharidin . ( 2011a ) hierarchical clustering of microarray data ( heat map ) from the analysis of arabidopsis thaliana genes which changed expression 2 , 10 , 24 hr after treatment with 200 m cantharidin . previously unpublished data from the experiments reported by bajsa et al . ( 2011a ) the phytotoxic trichothecene deoxynivalenol ( don ) produced by the plant pathogen fusarium graminearum had distinct effects on transcription in barley ( gardiner et al . , 2010 ) . transcripts for abc transporters , udp - glucosyltransferases , cytochrome p450s , and glutathione - s - transferases were up - regulated . biochemical studies supported the view that these upregulated genes were involved in detoxification of don . transcriptome responses to a particular compound can vary considerably , depending on duration and method of treatment and dose(s ) used . therefore , such experiments should be done with more than one dose , and sampling should be done at several times after exposure . since microarray methods do not provide quantitatively linear results , rt - qpcr ( reverse transcription - quantitative real - time pcr ) methods should be used to verify and obtain more accurate data on genes of interest identified with microarray methods ( e.g. , baerson et al . , 2005 ) . thus , extraction of rna from an entire plant , root , or shoot may dilute a more clear response that would have been obtained from the target tissue(s ) or cell type(s ) . analysis of biosynthetic pathways , in addition to individual genes , may provide clearer hints to the mode of action of a toxicant . for example , although there were no profound effects of an array of ergosterol synthesis - inhibiting fungicides on any of the genes encoding enzymes of the ergosterol synthesis pathway in saccharomyces cerevisiae , analysis of the pattern of effects on the entire pathway provided a signature that differentiated class i and ii ergosterol synthesis inhibitors from other fungicide modes of action ( kagan et al . , 2005 ) 2effects of class i ( a ) , ii ( b ) , and iii ( c ) sterol biosynthesis inhibitors , and a putative methionine biosynthesis inhibitor ( cyprodinil , d ) on expression levels of genes in the ergosterol pathway . standard errors are shown in a and b , and standard deviations are shown in c and d. genes are listed on the x - axis from left to right in the order in which they appear in the pathway . dashed horizontal lines on the graphs indicate the level of expression at which no change is seen relative to the control . arrows indicate gene(s ) encoding enzymes targeted by each inhibitor class . reproduced from kagan et al . ( 2005 ) effects of class i ( a ) , ii ( b ) , and iii ( c ) sterol biosynthesis inhibitors , and a putative methionine biosynthesis inhibitor ( cyprodinil , d ) on expression levels of genes in the ergosterol pathway . standard errors are shown in a and b , and standard deviations are shown in c and d. genes are listed on the x - axis from left to right in the order in which they appear in the pathway . dashed horizontal lines on the graphs indicate the level of expression at which no change is seen relative to the control . arrows indicate gene(s ) encoding enzymes targeted by each inhibitor class . reproduced from kagan et al . ( 2005 ) the whole transcribed genome microarray was a huge leap forward in transcriptome analysis ( redman et al . , 2004 ) . however , high - throughput , next generation sequencing of cdna from mrna or rna - seq offers another leap forward ( morozova and marra , 2008 ; wang et al . , 2009 ; marioni et al . , the number of copies of transcripts from different genes is determined by sequencing cdna fragments that are of sufficient length to identify the genes . these methods allow one to count directly the relative number of times a gene has been transcribed in a sample with statistical robustness . this method is much more precise than microarray methods , and it allows one to do transcriptomics experiments with plant species for which microarray chips do not exist . the dynamic range of this method is greater than 8,000-fold , whereas that for microarray is only up to a few hundred - fold ( wang et al . , 2009 ) . as a result , rna - seq methods are much more accurate for both highly and poorly expressed genes than is microarray . a downside of this method is that many millions of cdnas must be sequenced to determine definitive effects on weakly transcribed genes . however , this method provides the opportunity to obtain more accurate expression level estimates than can be obtained with microarray methods . until recently , the expense of this approach has been prohibitive , but in the past few years , the cost has become competitive with microarray . we have seen no papers that use this method to probe the mode of action of phytotoxins . there is a danger that rna - based data can result in false conclusions about the influence of a phytotoxin on proteins resulting from transcriptional effects . gene expression also is dependent on factors other than transcription , such as post - transcriptional processes , which can cause the protein levels to change independently of mrna . we are unaware of transcriptome information leading to the discovery of a target site of a phytotoxin , but there are numerous cases in which it has resulted in a better understanding the mode of action of phytotoxins ( e.g. , zhu et al . , 2008 ) . if a transcriptional pattern is too complex , determinning what gene(s ) might be associated with the primary target of the phytotoxin is challenging , even with rna - seq . as stated by duke et al . ( 2009 ) , transcriptional profiling can be useful in creating a list of candidate targets and pathways that is unmatched by any other technologies . this information can provide a foundation for the development of a hypothesis concerning the putative primary cellular target or targets of a phytotoxin . identification of proteins separated on two dimensional gels ( 2d page ) with mass spectrometric methods has allowed scientists to determine effects of phytotoxins on hundreds of proteins . there are thousands of proteins in plant cells , so this method is not as global as transcriptional analysis . nevertheless , the effects on relative abundance of proteins are closer to actual physiological effects than are transcriptional responses . there are only a few papers on analysis of proteome responses of plants to herbicides , and only a couple of papers are available that use this method to probe effects of putative allelochemicals and natural phytotoxins . 2012 ) examined the effects of low and high doses of the herbicides paraquat ( a psi energy diverter ) , diuron ( a psii inhibitor ) , and norflurazon ( a phytoene desaturase inhibitor ) on the proteome of the green alga chlamydomonas reinhardtii . significant effects ( both up- and down - regulation ) were found on 149254 proteins , depending on the herbicide . for example , although norflurazon had no effect on the abundance of its target enzyme , phytoene desaturase , other enzymes of the plastidic isoprenoid pathway ( e.g. , 1-deoxy - d - xylulose 5-phosphate synthase and geranylgeranyl reductase ) were upregulated , as were some enzymes of the tetrapyrrole pathway . in the case of diuron , the amounts of the d1 protein , its molecular target , and the light harvesting complex protein were increased , whereas levels of some other proteins involved in photosynthetic electron transport were decreased . heat map analysis of effects of the three herbicides on proteins associated with photosynthesis showed distinct patterns of up- and down - regulation associated with each of the herbicides ( fig . 3heat map showing proteins functionally related to the photosynthetic machinery which were significantly changed in chlamydomonas reinhardtii in response to paraquat ( pq ) , diuron ( dr ) , and norflurazon ( nf ) at high ( h ) and low ( l ) concentrations . statistical evaluation was performed with g - test , fold changes are presented as log 2 values . reproduced from nestler et al . ( 2012 ) with permission heat map showing proteins functionally related to the photosynthetic machinery which were significantly changed in chlamydomonas reinhardtii in response to paraquat ( pq ) , diuron ( dr ) , and norflurazon ( nf ) at high ( h ) and low ( l ) concentrations . statistical evaluation was performed with g - test , fold changes are presented as log 2 values . reproduced from nestler et al . ( 2012 ) with permission proteomic analysis of roots of medicago truncatula treated with als - inhibiting herbicides flumetsulam and metsulfuron methyl revealed more proteins were affected in meristematic ( 81 ) than in non - meristematic ( 51 ) tissues ( holmes et al . , 2006 ) . there were two trends : 1 ) increased accumulation of proteins involved in cell division and redox mediation in meristematic than in other tissues , and 2 ) increases in proteins involved in pathogen responses and decreases in metabolic proteins in all tissues . kelley et al . ( 2006 ) found four proteins to be strongly affected by the two auxinic herbicides dicamba and clopyralid in soybeans . although it was not detected by 2-d gel , this protein was probed by immunoblot analysis since the transcription of the gene was strongly upregulated ( see transcriptomics section ) . they were tentatively identified as malate dehydrogenase ( md ) , gamma - glutamyl - hydrolase , and superoxide dismutase ( sod ) . sod is a stress protein that would be expected to be upregulated by phytotoxins for many modes of action , and md upregulation has been associated with general plant stress . the upregulation of the gh3 gene and protein was suggested as a biomarker for auxinic herbicide damage . sublethal treatments of vitis vinifera with the protoporphyinogen oxidase ( ppo)-inhibiting herbicide flumioxazin caused abundance of 33 proteins to change ( castro et al . , 2005 ) . the herbicide up - regulated proteins associated with pathogenesis , oxygen stress , and photorespiration , whereas levels of some enzymes of carbon fixation and sugar metabolism were reduced . the authors speculated that reductions of some proteins may have been due to enhanced degradation of the proteins , a process that has been found to be an indirect effect of ppo inhibitor herbicides . proteomic analysis of maize revealed that the microtubule - disrupting herbicide amiprophos methyl changed the amount of 28 proteins , allowing the detection of 15 additional proteins and disappearance of 13 proteins with 2-d gels ( wang et al . , 2011 ) . some of the proteins affected in the root , leaves , and mesocotyl were a cold acclimation protein , ubiquitin , a ubiquitin - like protein , maturase k , and a cytochrome p450 monoxygenase in the mesocotyl , ferredoxins and 2,4 , dienoyl - coa reductase of the root , and an atp - dependent protease . they concluded that the profile of affected proteins could be used as a marker for phytotoxins acting through this mode of action . the effects of the long chain fatty acid elongase - inhibiting herbicide dimethenamid and the herbicide safener cloquintocet - mexyl on the proteome of roots , leaves , and coleoptiles of triticum tauschii were analyzed by zhang et al . proteins increased by the safener were involved predominately in xenobiotic detoxification , mainly in the coleoptiles and root . no multidrug resistance - associated proteins ( mrp ) were detected by 2-d gels , but enhanced safener - induced transcription of one mrp was found by rt - qpcr . the effects of crude extracts of allelopathic mexican plant species on the proteome of bean and tomato roots revealed alterations in only 11 to 16 proteins , depending on the source of the extract and test plant species ( cruz - ortega et al . , 2004 ) . in these relatively early studies , only an -amylase inhibitor - like protein and glutathione s - transferase were identified as being affected . both of their levels increased , but this provided few clues to understanding the physiological mechanisms of allelochemical - induced stress . we have not found any studies on the effects of single allelochemicals on the proteome . ( 2011 ) identified 25 differentially expressed proteins in a chinese medicinal achyranthes species treated with growth - promoting extracts of soil in which the same species was growing . proteins associated with stress and secondary plant metabolism , signal transduction , synthesis and degradation of nutritive material , as well as synthesis and degradation of nucleic acids and protein were affected . this paper did not identify a target site , but the actual active compounds in the extract also were not identified . proteomics , using ms analysis of 2-d gel spots is not at the level of precision as transcriptomics , whether using microarrays or second generation sequencing . for example , the transcriptional clue that the gh3 gene is up - regulated by auxinic herbicides could not verified by 2-d gel analysis , but was verified with a more sensitive immunochemical method ( kelley et al . , 2006 ) . the problems with proteomics outlined by zhang and riechers ( 2008 ) still largely exist . they include the lack of a template - based replication process such as pcr for rna . this means that visualizing and analyzing low abundance proteins from gels is impossible with the methods that have been used . reproducibility is a problem with 2-d gels ( abdallah et al . , 2012 ) . additionally , proteomics is limited if gene or transcript information is not available to identify the sequenced peptides . the complexity of posttranslational modification of proteins and its regulation is a difficulty that is not encountered with rna . another drawback it that proteomics is limited to soluble proteins , excluding many membrane - bound proteins ( trimpin and brizzard , 2009 ) . in general , the correlation of transcriptomes and proteomes is not good , at least partly because actually translated rna is only a part to the total rna usually analyzed with transcriptomics methods . using only polysomal rna in transcriptional studies may reduce the lack of correlation ( skadsen and jing , 2008 ) . recently developed gel - free proteomics methods such as itrag ( isobaric tags for relative and absolute quantitation ) and other protein tagging methods coupled with lc - ms / ms may become powerful tools for tackling such problems and improving on 2-d gel - based proteomics ( bindschedler and cramer , 2011 ; abdallah et al . , 2012 ) . less complicated , lable - free , gel - free lc / ms methods also are available ( abdallah et al . , 2012 ) . we have found no literature using these newer proteomics methods to probe the mode of action of phytotoxins . compared to the number of genes and/or proteins in a single plant species , there are a much smaller number of metabolites with a molecular weight less than 500 , especially primary metabolites required for growth and development . if one includes secondary metabolites , all plant species combined have over 100,000 different compounds ( dixon , 2001 ) , but most plant species contain less than a thousand primary metabolites and perhaps as many secondary metabolites , some of the latter of which are often exclusive to a single or a closely related group of plant species . herbicides and other potent phytotoxins do not kill plants by interfering with secondary plant metabolism , so metabolomic approaches to understanding mode of action can concentrate on the relatively small number of primary metabolites . however , effects of phytotoxins on concentrations of some secondary compounds might be useful in generating a fingerprint for some modes of action . modern chromatographic separation instrumentation coupled with mass spectrometry and/or nmr allows the separation and tentative identification of most of these compounds with relative ease . one problem with this approach is that the concentrations of even primary metabolites can be orders of magnitude apart , and can vary dramatically between cell types and different tissues . some metabolic intermediates are under tight regulation and do not exceed very low concentrations , as they can be toxic to the plant at higher concentrations . examples of these toxic intermediates include sphingoid bases , protoporphyrinogen ix , and -ketobutyric acid ( abbas et al . , 2002 ; duke , 2012 ) . in this short review these details can be found in more extensive reviews dealing only with metabolomics ( e.g. , aliferis and chysayi - tokousbalides , 2011 ) . one approach to metabolomics is to generate one - dimensional ( 1d ) proton nmr ( h nmr ) spectral fingerprints of crude extracts , hoping that the resulting information can provide clues to the mode of action , by comparing with fingerprints of phytotoxins with known modes of action ( e.g. , ott et al . , 2003 ; aliferis and chysayi - tokousbalides , 2006 ) . for obvious reasons , this method is inferior to actually identifying and quantifying each compound . two - dimensional ( 2d ) nmr with correlation spectroscopy ( cosy ) analyses of crude extracts can provide more chemical data , but the method is inferior to chromatographic separation , followed by mass spectroscopy . lc - nmr has advantages over nmr alone , but it has limited detection limits compared to ms coupled with chromatography . use of both gc - ms and lc - ms / ms for the same samples has provided perhaps the most definitive information on metabolomics thus far generated for herbicide modes of action ( grossmann et al . , 2010 , 2012a , b ) . there have been relatively few studies that use metabolomic approaches to mode of action discovery or confirmation for phytotoxins . the review of aliferis and jabaji ( 2011 ) gives details about the use of metabolomics to profile modes of action of pesticides , including herbicides . the first use of nmr for fingerprinting the mode of action of herbicides by metabolite analysis was by aranibar et al . four modes of action were fingerprinted by using artificial neural networks for classification for h - nmr data from crude extracts of maize . this work was later expanded to 19 modes ( ott et al . , 2003 ) . the mode of action of the phytotoxin ( 5s,8r,13s,16r)-(1)-pyrenophorol was probed in avena sterilis by using a h - nmr fingerprinting of metabolites ( aliferis and chysayi - tokousbalides , 2006 ) . the fingerprint did not correspond to those of the herbicides diuron , glyphosate , mesotrione , norflurazon , oxadiazon , or paraquat . in lemna minor , they later claimed that this compound caused effects on the metabolome similar to that of glyphosate , and that their results suggested a similar mode of action ( aliferis et al . , 2009 ) , yet they did not check the dramatic elevation in shikimic acid , the telltale marker for inhibition of epsps , the target site of glyphosate ( duke et al . , 2003 ) . identification of individual metabolites would have confirmed whether or not this claim was correct . in metabolomic studies of the effects of glyphosate on a. thaliana using lc - pda , lc - ms , and gc - tof - ms ( bttcher et al . , 2008 ) and on lemna pausicostata using a combination of gc - ms and lc - ms / ms ( grossmann et al . , 2012b ) , large increases in shikimate and effects on amino acid profiles were the predominant early effects of glyphosate . ( 2008 ) showed that when a commercial formulation of glyphosate was used , the extracts contained significant amounts of formulation chemicals that would not have been discerned with methods that do not separate mixtures into their components . h- nmr analysis of complex mixtures without separation is rapid , but it does not provide clues to the mode of action if the compound profile does not match that of a phytotoxin with a known mode of action . ( 2006 ) used direct infusion , fourier transform ion cyclotron resonance mass spectrometry ( ft - icr / ms ) , another method that does not separate , identify , and quantify metabolites to discriminate between metabolite profiles of four modes of action in a. thaliana . compounds with the same mode of action gave similar results , however , there was overlap between responses to compounds with different modes of action . using gc - ms , kluender et al . ( 2009 ) examined the effects of the psii - inhibiting herbicide prometryn on the metabolites of the green alga scenedesmus vacuolatus . results over a 14-h time course showed development of impairment of energy metabolism associated with catabolic processes , and reductions in carbohydrate synthesis . perhaps the most complete study of the effects of phytotoxins on plant metabolomes is that of trenkamp et al . they examined the effects of glufosinate , glyphosate , sulcotrione , foramsulfuron , benfuresate , and an experimental cell wall biosynthesis inhibitor ae944 . analysis of polar metabolites revealed clear differences in profiles generated by these compounds with six different modes of action . for some herbicides , the results matched what would be expected from the mode of action of the herbicide ( e.g. , sulcotrione and ae944 ) , and in other cases there was no apparent connection between the effects on metabolites and the mode of action ( e.g. , benfuresate ) . since benfuresate acts on lipid synthesis , one might not expect to see effects on polar metabolites that would point to a mode of action . another example of the use of metabolomics to probe secondary effects of a phytotoxin is that of cheng et al . ( 2011 ) , who examined the metabolome of soybean in response to the herbicide lactofen . herbicides with this mode of action induce host defenses against disease in soybean ( duke et al . , 2007 ) . lc - ms analysis of the herbicide - induced metabolites led to the discovery to two new phytoalexins . ( 2007 ) examined the effects of the phytotoxic constituents of the roots of ligularia macrophylla on growth of lemna paucicostata . separately , using the chromatography - coupled ms metabolomics methods described in duke et al . ( 2010 , 2012a , b ) , the effects of the most active of these eremophilane compounds , 6-angeloyloxy-10-hydroxyfurnoeremophilane , on the metabolome of l. paucicostata were examined ( fig . this method generates relative concentration values for about 200 identified metabolites and 300 unknown compounds . results are compared by cluster analysis with a proprietary database of about 150 metabolic profiles of previously characterized standard compounds on l. paucicostata with about 60 different modes of action ( grossmann et al . , 2012b ) . strong increases in certain sugars , abscisic acid , l - dopa , and tryptophan , and decreases in allantoin , aminoallantoins , flavonoids , and cystathionine were observed in response to the furanoermophilane . 2010 ) found the metabolic profile of saflufenacil to correspond closely to those of ppo inhibitors . this mode of action was confirmed by in vitro enzyme assays and by determining that the compound causes rapid and dramatic increases in protoporphyrin ix in vivo.fig . nodes of metabolites indicate significance of changes at p < 0.01 ( dark ) , 0.01 < p < 0.05 ( middle ) , or 0.05 < p < 0.1 ( light ) levels . we thank klaus grossmann of basf se , limburgerhof , germany , charles cantrell , of usda , ars , oxford , ms , usa , and nicole christiansen of metanomics , berlin , germany for these previously unpublished data metabolite profile of lemna paucicostata treated with 6-angeloyloxy-10-hydroxyfurnoermophilane from ligularia macrophylla . nodes of metabolites indicate significance of changes at p < 0.01 ( dark ) , 0.01 < p < 0.05 ( middle ) , or 0.05 < p < 0.1 ( light ) levels . we thank klaus grossmann of basf se , limburgerhof , germany , charles cantrell , of usda , ars , oxford , ms , usa , and nicole christiansen of metanomics , berlin , germany for these previously unpublished data the same method was used in conjunction with physionomics to study the mode of action of cinmethylin , an older herbicide with an unknown mode of action ( grossmann et al . it is a structural analogue of the potent natural phytotoxin , 1,4-cineole ( romagni et al . , 2000 ) . in addition to other effects , an isa caused higher levels of tryptophan and tyrosine , while phenylalanine concentrations were strongly reduced . the tyrosine degradation product l - dopa ( 3,4-dihydroxyphenylalanine ) increased up to five - fold , and plastohydroquinone levels were reduced . these effects were similar to those of the hppd inhibitor topramezone , but differed in that the isa did not cause a decrease in isopentenyl pyrophosphate and tocopherol . cluster analysis of metabolite changes compared effects of phytoene desaturase , hppd , and non - mevalonate isoproenoid synthesis inhibitors which have some common effects with cinmethylin and the isa compounds ( grossmann et al . cinmethylin and two isas ( methiozolin and iso1 ) clustered distinctly differently from the other herbicide classes . feeding treated plants with 4-hydroxyphenylpyruvate ( 4-hpp ) , a direct tyrosine derivative , alleviated the growth inhibition(grossmann et al . tyrosine amino - transferase , the enzyme that converts tyrosine to 4-hpp , was inhibited in vitro by cinmethylin and the isos . this is the only paper of which we are aware that has used omics methods to discover a new molecular target site.fig . 5mode of action classification by cluster analysis of metabolite changes in lemna paucicostata 48 and 72 hr after treatment with herbicides affecting isoprenoid synthesis . phytoene desaturase ( pds ) , hydoxyphenylpyruvate deoxygenase ( hppd ) , and non - mevalonate isoprenoid synthesis inhibitor modes of action contrast with the results from cinmethylin , methiozolin , and iso1 ( see fig . 7 for structures ) . 6effects of tyrosine and downstream products of tyrosine amino transferase ( 4-hyroxyphenylpyruvate and homogentisate ) on the growth inhibition of lemna paucicostata by cinmethylin , methiozolin , and iso1 . from grossmann et al . ( 2012a ) with permission mode of action classification by cluster analysis of metabolite changes in lemna paucicostata 48 and 72 hr after treatment with herbicides affecting isoprenoid synthesis . phytoene desaturase ( pds ) , hydoxyphenylpyruvate deoxygenase ( hppd ) , and non - mevalonate isoprenoid synthesis inhibitor modes of action contrast with the results from cinmethylin , methiozolin , and iso1 ( see fig . 7 for structures ) . from grossmann et al . ( 2012a ) with permission effects of tyrosine and downstream products of tyrosine amino transferase ( 4-hyroxyphenylpyruvate and homogentisate ) on the growth inhibition of lemna paucicostata by cinmethylin , methiozolin , and iso1 . from grossmann et al . ( 2012a ) with permission a more recent paper ( grossmann et al . , 2012b ) provides considerable metabolomic information using their protocols about a number of known modes of action , as well as new information about a phytotoxic phenylalanine analog ( phe1 ) . changes in several metabolites , including increases in tryptophan levels , led these researchers to hypothesize that the compound inhibits iaa synthesis . supplying the plants with iaa and several intermediates of iaa synthesis reversed growth inhibition by phe1 to a great extent . the enzyme(s ) of iaa synthesis that are the most likely the target site(s ) have not yet been isolated . ( 2011 ) used the same method to probe the mechanism of action of the potent fungal phytotoxin ascaulitoxin aglycone , produced by the plant pathogen ascochyta caulina . the metabolic profile did not correspond to any of the 60 modes of action that have been elaborated with this method . however , it caused distinct changes in amino acid contents , which indicated that it might inhibit conversion of pyruvate to alanine or the synthesis and/or interconversion of glutamate / glutamine and aspartate / asparagine . physionomics is based upon profiles of physiological responses to effectors ( grossmann , 2005 ; grossmann et al . , 2012b ) . grossmann ( 2005 ) first proposed the term physionomics , and he and his colleagues have used this approach successfully in conjunction with metabolomics to study the mode of action of several compounds ( see above ) . this procedure is similar to past approaches of herbicide discovery by companies for discovery of phytotoxin modes of action , however , the physiological profiles for different modes of action are quantified for clearer comparisons with other and unknown modes of actions with the methods of grossmann ( 2005 ; grossmann et al . scientists in the public sector have proposed batteries of physiological assays to identify the mode of action of phytotoxins ( e.g. , dayan et al . , 2000 ) . these physiological assays of the physionomics approach can provide a first clue to a mode of action that in many cases can eliminate the need for more complicated and expensive omics approaches.fig . 7physionomic profiles of four compounds that are apparently phytotoxic due to inhibition of tyrosine amino transferase . bioassay abbreviations : d , dark ; l , light ; vlcfa , very long chain fatty acid ; ros , reactive oxygen species . symptoms observed : a , tissue desiccation ; i , root growth inhibition ; n , necrosis of meristematic area ; wr , intensified green leaf pigmentation . reproduced with permission from grossmann et al . ( 2012a ) physionomic profiles of four compounds that are apparently phytotoxic due to inhibition of tyrosine amino transferase . bioassay abbreviations : d , dark ; l , light ; vlcfa , very long chain fatty acid ; ros , reactive oxygen species . symptoms observed : a , tissue desiccation ; i , root growth inhibition ; n , necrosis of meristematic area ; wr , intensified green leaf pigmentation . reproduced with permission from grossmann et al . omics approaches to mode of action discovery can only point the way to the molecular target of a phytotoxin . target sites can be partially validated through genetic means , such as determination that a defect in the gene encoding the putative target site generates the same phenotype , including the same omics profile . however , short - term blockage of a biochemical or signaling pathway by a phytotoxin is not likely to cause a similar omics profile as that caused by a genetic defect of the same target . one might expect that if the mutation is not lethal , compensatory mechanisms would have developed . with a. thaliana , this includes characterized classical mutants generated by the mutagen ethyl methane sulfonate ( ems ) ( http://www.arabidopsis.org/ ) and t - dna insertion lines ( krysan et al . , 1999 ) . however , ultimately , the target site must be validated by showing that it binds the phytotoxin and is inhibited in vitro . with some natural phytotoxins , this can be complicated because the compound is a protoxin ; that is , it must be metabolically activated to form the active inhibitor in vivo . examples of this are the microbial phytotoxin hydantocidin , which must be phosphorylated to inhibit adenylosuccinate synthase ( cseke et al . , 1996 ) and bialaphos , the product of a soil microbe , which must be degraded to phosphinothricin to inhibit glutamine synthetase ( wild and zeigler , 1989 ) . the strongest validation of a molecular target site of a phytotoxin is a genetic change in the gene of the molecular target site that renders that protein and producing plant resistant . omics technologies lend themselves to shotgun experiments with no clear biological rationale , unlike one might have for an experiment with a clear biomarker that predicts cause and effect . the amount of data and complexity of omics studies require complicated analytical capabilities that often do not provide clear answers to biological questions . even in the most advanced omics work done in the medical field , these problems have been very challenging ( institute of medicine , 2012 ) . as pointed out by jamers et al . ( 2009 ) , the gap between global nucleic acid - based omics ( genomics and transcriptomics ) and other forms of omics is large in terms of analyzing the complete array of proteins , metabolites , and/or physiological effects . furthermore , even though modern instrumentation and software has reduced the cost and labor involved in generating and analyzing the huge data bases that are required for rigorous fingerprinting of known modes of action , such an enterprise is still larger than most independent laboratories can bear . nevertheless , omics methods offer greater insight into all of the effects of a phytotoxin on the biochemistry and genetics of a target plant . in addition to seeing how the compound injures the plant , one can answer questions such as mechanisms of defense against the compound ( e.g. , baerson et al . , 2005 ) , potential secondary target sites ( e.g. , zhu et al . , 2008 ) , and sublethal effects on plant constituents . considering that these technologies are only about a decade old , we have only begun to fully employ them to answer questions related to natural phytotoxins and chemical ecology .

peters anomaly is characterized by a central corneal opacity with corresponding defects in the posterior stroma , descemet 's membrane , and endothelium . we present 2 cases that showed corneal opacity when examined by topical endoscopic imaging ( tei ) . case 1 was a 20-day - old neonatal female who had a central corneal opacity in the left eye . tei showed that the iris stroma was adhered toward the back of the opacified cornea . case 2 was a 4-month - old male who had a bilateral corneal opacity . tei revealed that both a keratolenticular adhesion and a surrounding iridocorneal adhesion were observed behind the area of corneal opacity . the patient was diagnosed as having peters anomaly with persistent fetal vasculature . this study demonstrates that tei is a novel method capable of looking into an eye from only a small area of the clear cornea .

this anomaly is characterized by a central corneal opacity with corresponding defects in the posterior stroma , descemet 's membrane , and endothelium [ 2 , 3 , 4 , 5 ] . ultrasound biomicroscopy is a useful tool for detecting associated structural abnormalities such as keratolenticular or iridocorneal adhesions . however , without the patients cooperation , it can be difficult to obtain a desirable image when using this method . topical endoscopic imaging ( tei ) is a new tool that can be used to examine intraocular findings through a clear cornea . here , we present 2 cases that showed corneal opacity when examined by tei . after the parents of the patients had been given a written explanation of the study , they all provided written informed consent . a 20-day - old neonatal female was referred to our department because of corneal opacity . although her right eye ( od ) was normal , her left eye ( os ) showed a central corneal opacity ( fig . the anterior chamber structures of the os could be partially visualized through the clear area . fundus examination showed a normal appearance of the od and os except for the superior periphery , which was difficult to observe because of the corneal opacity . thus , to examine the anterior chamber in detail , we performed tei with a previously reported method [ 7 , 8 ] . briefly , the authors used a rigid endoscope with an otoscope 6.0 cm in length and an outer diameter of 4 mm ( 1215aa ; karl storz , tuttlingen , germany ) with a crescent - shaped illuminating tip . a xenon lamp ( xenon nova 175 ; karl storz ) the endoscope was connected to a digital camera utilizing a 400,000-pixel charge - coupled device image sensor unit , which was connected to a monitor in turn . the patient was placed on a bed in the supine position , and topical anesthetic was instilled into the os . a spatula was placed between the eyelids , and hydroxyethyl cellulose solution was applied on the corneal surface to protect it and to create an interface that would improve the quality of the image . the endoscope was placed in proximate contact with the cornea and directed such that the angle could be observed . as shown in figure 1b , the iris stroma was adhered toward the back of the opacified area of the cornea , which confirmed the diagnosis . a 4-month - old male was referred to our department because of a bilateral corneal opacity . our examination demonstrated the presence of a bilateral central corneal opacity and extremely shallow anterior chambers , which made it impossible to observe the fundus in both eyes ( fig . the intraocular pressure measured was 8 mm hg in the od and 9 mm hg in the os . ultrasonographic investigations showed the presence of short axial lengths ( od : 16.0 mm ; os : 16.2 mm ) and funnel - shaped retinal detachment . when tei was performed via the clear peripheral cornea , both a keratolenticular adhesion and a surrounding iridocorneal adhesion were observed behind the area of corneal opacity ( fig . a 20-day - old neonatal female was referred to our department because of corneal opacity . although her right eye ( od ) was normal , her left eye ( os ) showed a central corneal opacity ( fig . the anterior chamber structures of the os could be partially visualized through the clear area . fundus examination showed a normal appearance of the od and os except for the superior periphery , which was difficult to observe because of the corneal opacity . thus , to examine the anterior chamber in detail , we performed tei with a previously reported method [ 7 , 8 ] . briefly , the authors used a rigid endoscope with an otoscope 6.0 cm in length and an outer diameter of 4 mm ( 1215aa ; karl storz , tuttlingen , germany ) with a crescent - shaped illuminating tip . a xenon lamp ( xenon nova 175 ; karl storz ) the endoscope was connected to a digital camera utilizing a 400,000-pixel charge - coupled device image sensor unit , which was connected to a monitor in turn . the patient was placed on a bed in the supine position , and topical anesthetic was instilled into the os . a spatula was placed between the eyelids , and hydroxyethyl cellulose solution was applied on the corneal surface to protect it and to create an interface that would improve the quality of the image . the endoscope was placed in proximate contact with the cornea and directed such that the angle could be observed . as shown in figure 1b , the iris stroma was adhered toward the back of the opacified area of the cornea , which confirmed the diagnosis . a 4-month - old male was referred to our department because of a bilateral corneal opacity . our examination demonstrated the presence of a bilateral central corneal opacity and extremely shallow anterior chambers , which made it impossible to observe the fundus in both eyes ( fig . the intraocular pressure measured was 8 mm hg in the od and 9 mm hg in the os . ultrasonographic investigations showed the presence of short axial lengths ( od : 16.0 mm ; os : 16.2 mm ) and funnel - shaped retinal detachment . when tei was performed via the clear peripheral cornea , both a keratolenticular adhesion and a surrounding iridocorneal adhesion were observed behind the area of corneal opacity ( fig . townsend et al . have subdivided peters anomaly into the following three types : ( i ) central corneal leukoma only ; ( ii ) central corneal leukoma and keratolenticular adhesion , and ( iii ) central corneal leukoma associated with axenfeld - rieger mesodermal dysgenesis . case 1 showed an iridocorneal adhesion , which corresponds to type i , while case 2 displayed bilateral corneal opacity with iridocorneal and keratolenticular adhesions , which matches type ii . case 2 also showed bilateral funnel - shaped total retinal detachment and a short axial length , assuming peters anomaly complicated with persistent fetal vasculature , as described previously . in the present cases , we employed the widely used otoscope to perform tei . utilization of this method makes it possible to easily visualize the anterior segment structure via a small clear area of the cornea [ 7 , 8 ] . when tei was used to examine case 1 , a small iridocorneal adhesion positioned toward the back of the corneal lesion was observed . in contrast , a prior ultrasound biomicroscopy examination of the same case could not detect this adhesion . moreover , utilization of this method in case 2 revealed a partially formed and extremely narrow anterior chamber with iridocorneal and keratolenticular adhesions . therefore , this study demonstrates that tei is a novel method that is capable of looking into an eye from only a small area of the clear cornea , although the image obtained with this system is a monocular image and its resolution is restricted by the performance of the charge - coupled device . in addition , this technique may also be helpful when investigating congenital abnormalities in the anterior chamber , angle , iris , and lens , especially in patients unable to remain in a sitting position .

dual - energy computed tomography ( dect ) simultaneously acquires images at two x - ray energy levels , at both high- and low - peak voltages ( kvp ) . the material attenuation difference obtained from the two x - ray energies can be processed by software to analyze material decomposition and to create additional image datasets , namely , virtual noncontrast , virtual contrast also known as iodine overlay , and bone / calcium subtraction images . dect has a vast array of clinical applications in imaging cerebrovascular diseases , which includes : ( 1 ) identification of active extravasation of iodinated contrast in various types of intracranial hemorrhage ; ( 2 ) differentiation between hemorrhagic transformation and iodine staining in acute ischemic stroke following diagnostic and/or therapeutic catheter angiography ; ( 3 ) identification of culprit lesions in intra - axial hemorrhage ; ( 4 ) calcium subtraction from atheromatous plaque for the assessment of plaque morphology and improved quantification of luminal stenosis ; ( 5 ) bone subtraction to improve the depiction of vascular anatomy with more clarity , especially at the skull base ; ( 6 ) metal artifact reduction utilizing virtual monoenergetic reconstructions for improved luminal assessment postaneurysm coiling or clipping . we discuss the physical principles of dect and review the clinical applications of dect for the evaluation of cerebrovascular diseases .

the concept of dual - energy computed tomography ( dect ) is based on the attenuation differential of various materials when simultaneously exposed to low- and high - energy x - rays . this reflects the difference in material interaction with low- and high - energy photons and resultant changes in compton scatter and photoelectric effects . compton scatter ( scattering of x - rays with little absorption ) predominates at the 120140 peak kilovoltage ( kvp ) energy range . in contrast , the photoelectric effect ( photon absorption ) is specific to an element and equates to the energy required to eject a k - shell electron . the photoelectric effect is proportional to the cube of the atomic number and inversely proportional to the incident photon energy ( z / e ) . conventional ct utilizes a single polychromatic x - ray tube with a peak energy of approximately 120 kvp and produces images with high signal - to noise ratio ; however , clear discrimination between different high attenuation materials such as iodine , hematoma and bone , or calcium can sometimes be difficult and may require additional acquisitions such as a noncontrast scan . one method employs two orthogonal x - ray tubes set at different kvp levels with two separate detectors . the second method uses rapid kvp switching from a single x - ray source and a detector composed of two scintillation layers . in both methods , the attenuation of fundamental materials is discernibly different from one another such as cerebrospinal fluid , brain parenchyma , hematoma , iodine and , calcification . a plot of the behavior of the energy attenuation on both x - ray tubes results in a physiological line reflecting the graduated increase in expected hounsfield units in each voxel with higher density material . presence of iodine within voxel changes the attenuation profile significantly and results in an upward deviation from the physiological line , and the gradient of the slope corresponds to the iodine concentration within the voxel where higher iodine concentration results in a larger gradient [ figure 1 ] . this change in attenuation profile is reflective of the binding energy of the k - shell electron ( k - edge ) of iodine which is 33.2 kev that is closer to the mean energy of the lower energy x - ray tube on a dect scanner , which is typically set as either 80 kv or 100 kv . this translates to nearly a two - fold increase in the attenuation level of iodine on 80 kv images compared to the 140 kv images . pathologies , which result in increased iodine concentration from either extravasation and/or neovascularity , will exhibit this unique dual energy property . complex algorithms have been developed to analyze the dect data using material decomposition algorithms based on the dual energy properties of material to differentiate brain parenchyma , hemorrhage , and iodine . standard multiplanar reconstructions are created on the weighted average image ( weighting factor of 0.4 ) , which resembles the conventional 120-kvp image of single - energy ct [ figure 2 ] . in addition , a virtual noncontrast ( vnc ) image can be created by the removal of iodine with no additional radiation exposure , closely mimicking the noncontrast image obtained from single - energy ct . virtual contrast ( vc ) image , also known as iodine overlay , can also be generated to enhance depiction of iodine contrast concentration and distribution . when necessary , additional bone and calcium subtraction images can be generated to allow better depiction of vessel lumens that would otherwise be obscured by bone or calcified atheromatous plaque . virtual monoenergetic images are useful to reduce the artifact surrounding metallic aneurysm coils or clips . principle of material decomposition algorithm is based on the different attenuation profiles of brain parenchyma , hematoma , and iodine . cerebrospinal fluid , brain parenchyma , and hematoma show similar hounsfield unit value on both 80 and 140 kv images . in contrast , iodine shows a two - fold increase in hounsfield unit value on 80 kv compared with 140 kv . the gradient of the slope ( red arrow ) correlate with the iodine concentration and increased gradient reflect greater concentration of iodine . dual - energy computed tomography angiogram at 80 kvp ( a ) , 140 kvp ( b ) and weighted average image ( c ) simulating a 120 kvp image . differences in the hounsfield unit attenuation between the images are illustrated by the hounsfield unit measurements within the left middle cerebral artery . the lowest energy 80 kvp image ( a ) shows greatest hounsfield unit as the energy level is closer to the k - edge of iodine . in contrast , the highest energy 140 kvp image ( b ) shows lowest hounsfield unit . the presence of contrast extravasation on ct angiography ( cta ) within a parenchymal hemorrhage , also known as the spot sign , has been shown to be associated with hematoma expansion and correlated with poor prognosis . the accurate depiction of a spot sign(s ) , therefore , potentially serves as a rapid noninvasive biomarker for higher risk hematomas and may directly influence decisions for medical or surgical management . currently , four phase ii randomized trials are underway to investigate the efficacy of early treatment with either recombinant activated factor vii or tranexamic acid in patients at a high risk for hematoma growth as depicted by the presence of a spot sign on ct angiogram ( stop - it [ clinicaltrials.gov nct00810888 ] , spotlight [ clinicaltrials.gov nct01359202 ] , traige [ clinicaltrials.gov nct02625948 ] , and stop - aust [ clinicaltrials.gov nct01702636 ] ) . dect improves the detection of subtle contrast extravasation points by increasing the conspicuity of iodine through the construction of a vc image [ figure 3 ] . this method reduces radiation dose in comparison to conventional dynamic single - energy ct techniques , which relies on separate multiphase acquisitions ( noncontrast , arterial , and delayed / washout phases ) to accurately depict active contrast extravasation . similarly , dect vc imaging can also be applied for the detection of active bleeding in other types of extra - axial hemorrhage . three examples of cerebral hematoma with active hemorrhage as depicted by a positive spot sign(s ) ( arrows ) on dual - energy computed tomography angiogram and virtual contrast images . first row : a 78-year - old male with known cerebral amyloid angiopathy ( a - d ) . second row : a 50-year - old female with poorly controlled hypertension ( e - h ) . third row : a 26-year - old male with amphetamine abuse ( i - l ) . the early recognition of hemorrhagic transformation of ischemic stroke is crucial ; however , accurate interpretation can be challenging due to residual parenchymal staining from extravasated iodine contrast during preceding diagnostic or neurointerventional procedures . dect vc imaging can again overcome this problem by facilitating accurate differentiation of iodinated contrast from hemorrhage with high sensitivity and specificity . dect vc images may be acquired from a noncontrast - enhanced ct head or a ct angiogram study , the latter is particularly useful where there is a need to assess vessel recanalization . iodine staining should be clearly depicted on the vc image but subtracted on the vnc image , and interpretation of both sets of postprocessing images may improve diagnostic confidence [ figure 4 ] . noncontrast computed tomography of the head ( a ) shows a focal intraparenchymal hyperdensity in the right lenticular nucleus in a patient following failed clot retrieval for the right middle cerebral artery stroke . dual - energy computed tomography cerebral angiogram virtual noncontrast image ( b ) shows subtraction of the right lenticular nucleus hyperdensity ( arrows ) . virtual contrast image ( c ) confirms the hyperdensity ( arrows ) that represents contrast staining . the presence of acute intracerebral hemorrhage has the potential to mask an underlying lesion such as a neoplasm or metastases . this ability to differentiate between hematoma and iodine may improve the detection of lesions that accumulate iodine contrast due to a pathologic breakdown in blood dect vc image has been shown to be significantly superior to conventional single - energy or weighted average dual energy image for the detection of lesional hemorrhage . improved contrast resolution from the vc image is particularly useful in the depiction of focal tumoral enhancement from regional intra- or peri - tumoral hemorrhage [ figure 5 ] . a 50-year - old woman with melanoma metastasis . single - energy noncontrast computed tomography image ( a ) shows acute intraparenchymal hemorrhage in the right frontal lobe with a suspicious juxtacortical lesion ( arrow ) which is difficult to differentiate from surrounding acute hemorrhage . computed tomography cerebral angiogram ( b ) again depicts subtle enhancement of the suspected juxtacortical lesion ( arrow ) . virtual contrast image ( c ) confirms the presence of a juxtacortical lesion ( arrow ) . concurrent magnetic resonance imaging brain fluid - attenuated inversion recovery ( d ) , pre- and post - contrast t1-weighted images ( e and f ) confirms the presence of an enhancing juxtacortical lesion . assessment of head and neck vascular anatomy with ct angiogram or venogram is often obscured by calcified atheromatous plaque or beam hardening artifact at the skull base [ figure 6 ] , which often proves problematic in assessing crucial vessels for pathology . using material decomposition analysis , dect postprocessing software allows automated subtraction of osseous or calcified plaques for optimal depiction of the vessel lumen . this technique offers a wide range of clinical applications , in particular for the evaluation of carotid plaque morphology and quantification of luminal stenosis . dect can also be used to characterize the material composition of atheromatous plaques and subtraction of calcium deposits within plaque lesions to improve the assessment of luminal caliber [ figure 7 ] . dual - energy computed tomography venogram with bone subtraction images ( a - c ) shows extensive right dural venous sinus thrombosis involving the distal transverse sinus , sigmoid sinus , and proximal internal jugular vein ( arrows ) . dual - energy computed tomography venogram without bone subtraction images ( d - f ) provided for comparison showing the dural venous thrombosis is less conspicuous , especially at the skull base ( arrows ) dual - energy computed tomography neck angiogram axial images ( a - d ) and with automatic hard plaque subtraction ( e - h ) shows moderate - to - high grade stenosis of the right proximal internal carotid artery by hard plaque ( arrow ) . note the automatic hard plaque subtraction images ( e - h ) better depict the degree of luminal stenosis than standard axial images ( a - d ) . rotating maximum intensity projections image ( i ) and with automatic hard plaque subtraction ( j ) shows clear visualization of the internal carotid artery which is otherwise obscured by the hard plaque ( arrow ) . imaging features of the atheromatous plaque that are often considered important in the risk assessment for stroke include the presence of severe stenosis [ figure 8 ] , plaque ulceration , and vasa vasorum enhancement . subtle plaque ulceration may be obscured from view by adjacent hard calcified plaque , and automated calcium subtraction improves visual assessment [ figure 9 ] . although carotid digital subtraction angiography remains the gold standard for the quantification of internal carotid artery stenosis , cta has become the more popular and accessible imaging modality . recent data have shown that dect - automated plaque and bone removal images with maximum intensity projections improve the accuracy of cta and more closely correlate with digital subtraction angiography compared to standard single - energy cta . an interpretation pitfall may be encountered in high grade or critical stenotic lesions when subtracted images can overestimate the severity of stenosis . this can be avoided with careful evaluation of both subtracted and unsubtracted dect images while correlating with standard reconstructions . dual energy - computed tomography neck angiogram axial images ( a - d ) and with automatic hard plaque subtraction ( e - h ) shows high - grade stenosis of the left proximal internal carotid artery by a circumferential hard plaque ( arrow ) . note the automatic hard plaque subtraction image overestimated the degree of stenosis when compared to the standard axial image . rotating maximum intensity projections image ( i ) and with automatic hard plaque subtraction ( j ) shows the proximal internal carotid artery which is otherwise obscured by the hard plaque ( arrow ) . a 38-year - old female with acute right middle cerebral artery ischemic stroke . dual - energy computed tomography neck angiogram with automatic hard plaque subtraction uncovering a fissured soft plaque flanked by adjacent calcified hard plaque . ( a - d ) show mixed morphology plaque at the carotid bifurcation with fissuring of the soft plaque arising from the posterior wall ( arrow ) . oblique sagittal reformation demonstrates the raised soft plaque from the posterior wall ( arrow , e ) with an irregular central fissure ( arrow , f ) . rotation maximum intensity projections with automatic bone and hard plaque subtraction clearly display the fissured soft plaque ( arrow , g ) . metal streak artifacts from aneurysm clips or coils greatly degrades image quality and limits accurate assessment of regional luminal enhancement . streak artifacts result from both beam hardening and photon starvation , and the severity of the artifact is dependent on the thickness and composition of the metal alloy . conventional ct techniques have attempted to reduce metallic artifacts by altering gantry angulation , multidetector scanning techniques , iterative reconstruction , and helical scanning with a 3d denoising filter ; however , the overall impact on improving image quality has remained minimal . postprocessing of dect angiography data allows extraction of the virtual monoenergetic images at the arbitrary photon energies ranging from 30 kev to 130 kev . the hounsfield unit ( hu ) values within the voxel on the monochromatic energy image reflect the attenuation from a beam of a single energy in contrast to the hu values derived from the polychromatic spectrum of a single - energy multidetector ct . the imaging processing algorithm is based on material decomposition and the specific mass attenuation of materials . theoretically , a true monochromatic image should contain no beam hardening artifacts because of the absence of spectral shifts . virtual monochromatic image , however , may still be hampered by scatter radiation or photon starvation although the noise level is significantly reduced compared with a single - energy multidetector ct polychromatic energy beam . hence , reducing beam hardening artifacts and providing more quantitatively accurate attenuation measurements are considered to be the main benefits of virtual monoenergetic images . at lower kev energy levels , virtual monoenergetic images show greater contrast enhancement of vasculature , but experience a greater degree of beam hardening and scatter effects [ figure 10 ] . at higher kev the trade - off between metal artifact reduction and contrast enhancement implies that optimal virtual monoenergetic images lie within the 90140 kev range with the median energy level of 113 kev selected as it has the lowest noise value in the area of maximum streak artifact [ figure 11 ] . imaging of aneurysm clips is more notably benefited from the reduced noise level ; however , the artifact from aneurysm coiling was less affected by kev changes . cta assessment and surveillance post aneurysm coiling or clipping are set to benefit from this technique , with the alternative imaging modality of magnetic resonance angiogram also frequently hampered by metal blooming artifact limiting detailed luminal assessment . representative dual - energy computed tomography angiogram images in a 49-year - old female post left middle cerebral artery aneurysm clipping . images ( a - h ) are reformatted monoenergetic images at 50 kev , 60 kev , 70 kev , 80 kev , 90 kev , 100 kev , 120 kev , and 130 kev . beam hardening artifact is reduced at 100 - 130 kev ; however , at higher kev , the contrast attenuation of the vessel is reduced . a 49-year - old female with a left middle cerebral artery bifurcation preoperative dual - energy computed tomography angiogram maximum intensity projections image ( a ) shows a left middle cerebral artery aneurysm at the m1/m2 junction ( arrow ) . immediate postoperative dual - energy computed tomography angiogram weighted average 120 kvp image ( b ) and virtual monoenergetic 113 kev image ( c ) show the aneurysm clip ( arrow head ) with improved metal artifact reduction and resolution of the adjacent vessel lumen using the virtual monoenergetic images ( arrow , c ) . knowledge of the principles and routine application of dect in clinical practice is invaluable in problem - solving for a diverse range of cerebrovascular diseases . the postprocessing steps can also be incorporated into certain routine ct protocols to improve diagnostic accuracy , especially in the clinical scenarios of carotid plaque assessments and post - treatment surveillance of cerebral aneurysms . as further dect techniques emerge from inception and development to implementation in clinical use practice

a 60-year - old female was treated for depression with the antidepressant paroxetine for 13 years . the patient had experienced clumsiness and mild rigidity in the left hand , and had agraphia and mild subjective memory complaints for 3 years prior to admission in our hospital . she experienced exacerbated depression that included worsened depressive mood , lowered motivation , and suicidal ideation without precipitating stressful life events for 2 years prior to admission , and although she had continued taking the antidepressant , these symptoms were not ameliorated by increasing the dose of paroxetine . following the development of myoclonus and pain in her left arm , we performed magnetic resonance imaging of her head , that revealed diffuse atrophy and right parietal lobe atrophy . the patient was ultimately diagnosed with corticobasal degeneration ( cbd ) . her left arm myoclonus and depression improved following levodopa administration . therefore , we concluded that the recurrent depression may have been induced by cbd .

a depressive state is frequently observed in patients with alzheimer - type dementia and mild cognitive impairment.13 it is known that depression is a risk factor for the development of alzheimer s disease.13 therefore , depression in middle - aged and older patients may be associated with organic brain changes . corticobasal degeneration ( cbd ) is a rare , progressive neurodegenerative disease characterized by progressive asymmetrical rigidity and apraxia.4 in addition , cbd can often present with complex cognitive difficulties and neuropsychiatric disturbances.4,5 symptoms of depression , apathy , or agitation can be subtle and are often overlooked as reactions to receiving a new diagnosis of parkinsonism.46 here , we report a case of exacerbated depression with cognitive impairment that was later diagnosed as cbd . after receiving a full description of the study , the patient provided written informed consent for this case to be published . a 60-year - old right - handed female with depression who was treated with the antidepressant paroxetine ( 20 mg / day ) for 13 years presented to our hospital . her mental status had remained relatively stable after primary treatment with the prescribed dose of paroxetine . the patient had experienced clumsiness and mild rigidity in her left hand and had agraphia and mild subjective memory complaints for 3 years prior to admission in our hospital . at another hospital , she was diagnosed with parkinson s disease ( pd ) and dementia and was administered anti - parkinsonian ( levodopa [ l - dopa ] , 200 mg / day ) and anti - dementia ( donepezil , 5 mg / day ) medications in addition to the antidepressant paroxetine ( 20 mg / day ) . she had been admitted to our hospital 2 years prior to the admission in the present case because she presented with exacerbated depression that included worsened depressive mood , lowered motivation , and suicidal ideation without any stressful life events . at admission , cognitive impairment as well as depression was observed , and thus , we performed a head magnetic resonance imaging ( mri ) , which revealed no cerebral atrophy ( figure 1a ) , including within the parahippocampal gyrus . dopamine transporter ( dat ) imaging showed no reduction in nigrostriatal dat accumulation . on the other hand , single - photon emission computed tomography neuroimaging showed a mild decrease in blood flow in the bilateral parietal lobes and posterior cingulate gyrus wedge . the neurobehavioural cognitive status examination ( cognistat ) indicated a normal range for naming and judgment , mild impairment of repetition , moderate impairment of similarity , and severe impairment of orientation , attention , comprehension , constructional ability , memory , and calculation . based on these findings , she was diagnosed with alzheimer s disease , and the anti - parkinsonian medication l - dopa decarboxylase was stopped and replaced with another anti - dementia drug , memantine , at a dosage of 10 mg / day . although the dosage of the antidepressant paroxetine was increased from 20 to 30 mg / day , the patient s depressive status was unchanged . after discharge , she developed myoclonus and pain in her left arm , and her depression worsened further . thus , she was reexamined , and a head mri revealed diffuse atrophy and right parietal lobe atrophy ( figure 1b ) . in addition , dat imaging showed a right - sided decrease in accumulation , and metaiodobenzylguanidine myocardial scintigraphy showed no deficit of accumulation . she was finally diagnosed with probable cbd according to armstrong s criteria for cbd.7 the patient was prescribed l - dopa ( 300 mg / day ) , and both her left arm myoclonus and depression improved . to date , the patient s cognitive function has further decreased , but her mental status has remained stable . although the depressive episode that had occurred 13 years prior to admission was not associated with cbd , it is likely that the depressive episode she had experienced 2 years prior to admission was caused by cbd because her depression did not improve with the increased dose of paroxetine and only improved after l - dopa was administered for cbd . based on her clinical course , the depressive episode probably recurred due to the development of cbd , despite being treated with an antidepressant ; therefore , this is the first case of recurrent depression caused by cbd . when depression is associated with neurological symptoms and cognitive dysfunction in elderly patients , cbd should be considered as a differential diagnosis . psychiatric symptoms in cbd have been shown to include depression , indifference , and irritability . it was reported that the total frequency of apathy , irritability , and disinhibition was 58% , whereas that of depression was 38%.8 eight of 36 autopsied cbd cases ( 22% ) had psychiatric symptoms , including behavioral dyscontrol ( 8.3% ) , depression ( 8.3% ) , compulsive behavior ( 8.3% ) , irritability ( 2.8% ) , and disinhibition ( 2.8%).9 another study showed that social withdrawal was the most common behavioral symptom in autopsy - confirmed cbd cases that fulfilled the diagnostic criteria of behavioral variant frontotemporal degeneration in life.10 disinhibition , stereotypy , and depression were the most frequent psychiatric symptoms , followed by aggression , apathy , self - centered behavior , social withdrawal , and euphoria.11 nonetheless , cognitive and psychiatric disturbances are common even in the early stages of the disease . although there are no guidelines for the treatment of depression in cbd , there are clear recommendations for the diagnosis of depression in pd.1214 antidepressant therapies include tricyclic antidepressants and selective serotonin reuptake inhibitors ( ssris ) ; however , it should be noted that ssris could worsen pd symptoms such as rapid eye movement sleep behavior disorder , periodic limb movement , and restless legs syndrome.15,16 there is some evidence suggesting the administration of dopamine agonists and mono - amine oxidase inhibitors for the treatment of depression in pd.12,15,16 pramipexole and selegiline have been suggested to have some antidepressant effects in addition to their motor effects.15,16 if the mood symptoms are only present during the off periods , patients might benefit from drugs targeting the motor symptoms.12 however , there is little evidence that l - dopa alone affects mood,16 although our case showed some efficacy at least . it can be challenging to distinguish early pd presenting with mild bradykinesia and dopamine - deficiency depressive symptoms from standard ssri - responsive depression associated with psychomotor retardation , which can be asymmetrical , while mild early pd motor signs can have only subtle asymmetry . dat imaging can help us distinguish between the two conditions to determine when depression and motor slowing should be treated with an ssri versus an l - dopa . the present case suggests that l - dopa may be effective for the treatment of depression associated with cbd , although there is little evidence to date regarding how to address psychiatric symptoms of cbd .

objectives : gastric endoscopic submucosal dissection ( esd ) is currently a standard procedure . esd enables en - bloc resection of large lesions , while inducing larger artificial ulcers to a greater extent than conventional procedures . several studies have reported that proton pump inhibitors ( ppis ) prevent delayed bleeding and expedite the artificial ulcer healing process . esomeprazole , an s - isomer of omeprazole , is reportedly one of strongest inhibitors of gastric acid secretion . previous studies have examined the effectiveness of esomeprazole . our goal was to verify the effects of esomeprazole on artificial ulcers in a prospective study.methods : a total of 185 patients underwent esd for gastric neoplasms at our hospital between january 2013 and june 2015 . among these 185 patients , 49 post - esd scar lesions were included in this prospective trial . first , 20 mg esomeprazole was orally administered to all subjects before and after the procedure . we then evaluated the delayed bleeding rate and ulcer scarring rates at 4 weeks and 8 weeks after the procedure by using a gastric ulcer stage system.results : there was one case of delayed bleeding ( 2.0% ) . regardless of helicobacter pylori infection status , ulcer scarring rates at weeks 4 and 8 were respectively 28.6% ( 14/49 ) and 98% ( 48/49).conclusions : our results suggest that oral administration of esomeprazole alone may be sufficient for prompt healing of artificial gastric ulcers induced by esd ( umin000009367 ) .

recently , endoscopic submucosal dissection ( esd ) has become a standard procedure for treating gastric intraepithelial neoplasms ( gastric adenoma and early gastric cancer ) . while esd allows for en - bloc resection of large lesions compared with endoscopic mucosal resection ( emr ) , the sizes of artificial ulcers induced by the procedure are proportionately large . kakushima et al . , reported that post - esd gastric ulcers heal within 8 weeks , regardless of size , location , status of helicobacter pylori infection , and extent of gastric atrophy . artificial ulcers theoretically remaining in the submucosal layer are thought to heal faster than peptic ulcers . several studies reported that proton pump inhibitors ( ppis ) are effective in preventing bleeding after the procedure and prompt healing of artificial ulcers . oh et al . reported that the initial ulcer size affects the ulcer healing status by ppi at week 4 of esd . if the size of the post - esd ulcer is larger than predicted , ppi administration alone may not be sufficient for treating ulcers . kato et al . reported that a combination of ppi and rebamipide was more effective than ppi alone for treating ulcers larger than 20 mm within 4 weeks of esd . esomeprazole , an s - isomer of omeprazole , is a new form of ppi and was reported to show stronger inhibition of gastric acid secretion than conventional ppis . the aim of this study was to evaluate the efficacy of esomeprazole alone for artificial ulcers induced by esd . a total of 185 patients underwent esd for gastric neoplasms at tsuchiura kyodo general hospital between january 2013 and june 2015 . among the 185 patients , written informed consent was obtained for entry into the trial . an oral dose of esomeprazole ( 20 mg ) the primary endpoints were the ratio of delayed bleeding and ulcer scarring rates at 4 and 8 weeks after the procedure ( figure 1figure 1study design . more than one week before the procedure , all patient were orally administered 20 mg / day esomeprazole ; the drug was also administered at 8 weeks after the procedure . ) . delayed bleeding was defined as clinically evident bleeding that required emergency endoscopic hemostasis and/or blood transfusion with a decline of more than 2 g / dl of hemoglobin . we evaluated ulcer scarring rates using a gastric ulcer staging system ( table 1table 1gastric ulcer stages classified using a 6-stage systemstageendoscopic definitiona1 ( active stage 1)ulcer that contains mucus coating , with marginal elevation because of edemaa2 ( active stage 2)mucus - coated ulcer with discretemargin and less edema than active stage 1h1 ( healing stage 1)unhealed ulcer covered by less than 50% regenerating epithelium with or without converging foldsh2 ( healing stage 2)ulcer with mucosal break but almost covered with regenerating epitheliums1 ( scar stage 1)red scar with rough epithelization without mucosal breaks2 ( scar stage 2)white scar with complete re - epithelization ) at weeks 4 and 8 after the procedure . more than one week before the procedure , all patient were orally administered 20 mg / day esomeprazole ; the drug was also administered at 8 weeks after the procedure . esds were performed using a conventional singe - channel endoscope with forward water - supply function ( gif - h260z or q260j , olympus , tokyo , japan ) . the endoscopy device in use was mainly the dual knife ( kd-650l , olympus ) . hyaluronic acid solution was injected into the submucosal layer for mucosal incision and physiological salt solution was used for submucosal dissection . the ulcer induced by esd was carefully examined , and any visible vessels were coagulated with hemostatic forceps ( fd-410l , olympus ) . all statistical analyses were performed using jmp version 10.0 software ( sas institute , cary , nc , usa ) . all data were expressed as the mean ( range , minimum maximum ) and the level of significance was set at p < 0.05 . data regarding the clinical and endoscopic features of the patients are shown in table 2table 2characteristics of patientssex ( male / female)39/10age ( years)mean 73.3 ( range , 5887)comorbiditieshypertension28diabetes mellitus5liver cirrhosis2hemodialysis2anticoagulant4antiplatelet drug13h . pylori . infection ( yes / no / unknown)20/22/7macroscopic typeprotruded type ( 0-i , 0-ii ) 28depressed type ( 0-iic)20flat type ( 0-iib)1location ( upper / middle / lower)6/17/26lesion ( adenoma / cancer)11/38tumor size ( mm)16.6 ( range 442)size of resected specimen ( mm)33.6 ( range , 1058)en - bloc resection46 ( 93.4%)operating time ( min)76.7 ( range , 15180 ) . h. pylori infection status was evaluated by serological testing , and some patients received h. pylori eradication therapy in this study . the mean tumor size was 16.6 ( range , 442 ) mm , and the mean size of resected specimens was 33.6 ( range , 1058 ) mm in diameter . en - bloc resection was attained in 93.4 ( 46/49)% of cases . no complications including perforation occurred during the trial except for one case of delayed bleeding . there was one case of delayed bleeding and the ratio was 2.0% ( 95% confidence interval ( ci ) : 2.16.1% ) . we defined ulcer scarring as s1/s2 stage and ulcer scarring rates at 4 and 8 weeks were 28.6% ( 95%ci : 17.842.4% ) and 98% ( 95%ci : 89.399.6% ) , respectively ( figure 2figure 2ulcer stage at weeks 4 and 8 after esd . ulcer scarring rates ( s1/s2 ) at weeks 4 and 8 of esd were 28.6% ( 95%ci : 17.842.4% ) and 98% ( 95%ci : 89.399.6% ) , respectively . ) . we assessed each background factor to identify correlations with ulcer scarring at week 4 after the procedure by multiplex logistic analysis , but no significant factors were identified ( table 3table 3factors involved in ulcer scarring at week 4odds ratiop - value95% confidence intervalage1.020.990.891.18location0.980.980.128.67size of resected specimen0.020.880.890.13operating time1.000.540.981.05hypertension0.230.190.021.96diabetes mellitus77600.99liver cirrhosis15520.99hemodialysis0.731.00anticoagulant0.000.990.0032.8antiplatelet drug0.470.750.025.87h . ulcer scarring rates ( s1/s2 ) at weeks 4 and 8 of esd were 28.6% ( 95%ci : 17.842.4% ) and 98% ( 95%ci : 89.399.6% ) , respectively . in japan , gastric cancer is one of the most common cancers and was the second leading cause of cancer - related death among men and the third leading cause among women in 2013 . the incidence of early gastric cancer is higher in japan than in other countries . emr is a fast and simple procedure , but it is difficult to achieve en - block resection of lesions larger than 20 mm in diameter . the incidence of procedure related - complications such as perforation and bleeding is higher in cases treated by esd than in cases treated by emr . delayed bleedings occur in 05% of endoscopically treated patients . to prevent delayed bleeding , post - esd preventive coagulation is effective and oral intake of ppi , compared with histamine-2-receptor antagonist ( h2-ra ) , is thought to be highly effective . bleeding from ulcers is considered to be one of the most serious and challenging complications during and after esd . green et al . suggested that intragastric ph should be greater than six in order to allow for platelet aggregation and prevent platelet disaggregation . inhibitors of gastric acid secretion such as ppi and h2-ra are indispensable for ulcer healing and prevention of post - esd hemorrhage . ppis are more commonly used for treating post - esd ulcers than other therapies . furthermore , some authors reported the beneficial effects of combination of ppis and anti - ulcer agents . rebamipide , a mucosal protective antiulcer drug , was effective in the healing process of artificial ulcers . kato et al . reported that a combination of ppi and rebamipide was more effective than ppi alone for ulcers larger than 20 mm in diameter at week 4 of esd . inaba et al . reported that in patients treated with lansoprazole plus polaprezinc , ulcer healing was significantly faster , and the incidence of protrusion of the ulcer base was significantly lower than in the patients treated with lansoprazole alone . to evaluate the effectiveness of esomeprazole in ulcer healing , our patients were administered mono - therapy with esomeprazole . reported that 4 weeks of ppi was not sufficient for healing of large post - esd ulcers and concluded that 8 weeks of treatment were required . arai et al . reported that 2 weeks of ppi administration may be sufficient for ulcer healing . however , there is no consensus regarding the proper duration of ppi administration after esd . esomeprazole was developed as a single optical isomer of racemic omeprazole and has shown some pharmacological advantages . a higher oral bioavailability contributes to a greater degree of acid suppression compared with omeprazole . the lower interpatient variability is likely related to the unique metabolic pathway of the drug . furuta et al . reported a number of patients who were refractory to ppis . depending on the cyp2c19 genotypes , patients are grouped into three types : rapid metabolizer , intermediate metabolizer , and poor metabolizer . esomeprazole appears to be less dependent on cyp2c19 , and thus functions as a stronger gastric acid secretion inhibitor . there have been a few reports of the impact of esomeprazole on the healing of artificial ulcers . reported that s1 stage was achieved at week 4 of esd in 27.6% , and 38.7% of patients treated with esomeprazole plus rebamipide and omeprazole plus rebamipide , respectively . arai et al . compared the staging of post - esd ulcers at week in patients receiving esomeprazole plus rebamipide for 2 weeks and 4 weeks , and found that the number of patients with ulcers in the healing / scar stage was 20/6 and 28/5 , respectively . in these studies , patients received intravenous administration of 20 mg omeprazole for one or two days after the procedure . reported no significant differences between patients receiving intravenous and those receiving oral administration of esomeprazole with respect to amount of time for which mean intragastric ph was greater than 4 on day 1 or day 5 of treatment following administration with 20 or 40 mg . therefore , our results suggest that esomeprazole should be administered more than one week before endoscopic therapy . kakushima et al . reported that gastric ulcers induced by esd heal within 8 weeks , but remain in the healing stage for 4 weeks . in this trial , at week 4 , ulcers reaching the scarring stage were detected in 28.6% of cases irrespective of specimen size , with a delayed bleeding rate of 2.0% . this result may be related to the strong inhibitory potential of esomeprazole against gastric acid secretion , and the use of a proper concentration of esomeprazole during the procedure by pre - administering the agents for one week before endoscopic therapy . thus , oral esomeprazole administration alone may be sufficient for preventing delayed bleeding and for ulcer healing . our results suggest that single - agent therapy with esomeprazole can be used as a standard therapeutic approach for treating artificial gastric ulcers induced by esd . omission of intravenous administration of ppi may also reduce the burden of doctors , nurses , and pharmacists during patient admission .

it has been revealed that the wnt/-catenin signaling pathway plays an important role in the development of solid tumors and hematological malignancies , particularly in b - cell neoplasia and leukemia . in the last decade there have been made experimental approaches targeting the wnt pathway in chronic leukemia . in this paper we provide an overview about the current state of knowledge regarding the wnt/-catenin signaling pathway in chronic leukemia with special focus on therapeutic options and strategies .

chronic myeloid leukemia ( cml ) is a one of the myeloproliferative diseases and represents 1520% of all adult leukemia types . depending on clinical aspects and laboratory findings , cml can be divided in three phases . typically cml begins in the chronic phase , and over several years it progresses to the accelerated phase and finally to the blast crisis . by the introduction of the bcr - abl tyrosine kinase inhibitor ( tki ) imatinib and the second - generation tkis dasatinib and nilotinib , nevertheless , resistance and incompatibilities against tkis are well known , and a molecular remission is difficult to achieve . curing patients with cml chronic lymphocytic leukemia ( cll ) belonging to the indolent b - non - hodgkin lymphoma ( b - nhl ) is the most common leukemia in the adult . the treatment of cll mainly depends on the clinical stage of the disease , which is determined by the binet staging system ( a c ) . in the early stage a watch and wait strategy is recommended . in progress of cll , the application of fludarabine , cyclophosphamide , and rituximab ( fcr ) is one of the treatment standards in the first - line therapy of cll . further alternative therapeutic options , particularly in progress or relapse and under consideration of several clinical aspects , have been established . these include the application of bendamustin and rituximab ( r - benda ) , chlorambucil , and alemtuzumab . despite the progress made in developing effective chemotherapeutic regimes the wingless - int ( wnt ) signaling pathway plays an essential role in embryogenesis and proliferation , survival , and differentiation of hematopoietic stem cells ( hscs ) [ 3 , 4 ] . it represents a complex network with mechanisms of self - regulation through positive and negative feedback . there have been described a large number of wnt proteins that activate various pathways in cells categorized as canonical and noncanonical wnt pathways . in this paper primarily the wnt/-catenin signaling pathway is focused . influencing the expression of genes involved in cell proliferation , differentiation and apoptosis are primarily due to the activation of the canonical -catenin and lymphoid enhancer factor ( lef)/t - cell factor ( tcf ) pathway . wnt proteins mainly stabilize cytoplasmic -catenin that has two essential functions : it is a very important element of many intracellular signaling pathways , including the wnt pathway , but it also takes part in creating intercellular adhesive junctions . signaling through -catenin a number of proteins such as adenomatous polyposis coli ( apc ) , axin , glycogen synthase kinase 3 ( gsk3 ) , casein kinase alpha ( ck1 ) , and -catenin form the destruction complex [ 8 , 9 ] . in the absence of an activating signal , phosphorylation of -catenin by gsk3 acting in conjunction with apc and axin causes -catenin to interact with the ligase -transducin repeat - containing protein ( -trcp ) which results in its ubiquitination and degradation , executed by proteasome . here , the transcription factor of the lef / tcf family , together with other proteins such as groucho , binds to dna and inhibits gene expression as a transcriptional repressor ( figure 1 ) . these are secreted glycoproteins that act as ligands for membrane receptors belonging to the frizzled ( fzd ) family of proteins and their coreceptors lipoprotein receptor - related protein 5 and 6 ( lrp 5 and 6 ) . consequently the protein disheveled ( dvl ) thereby , activity of gsk3 is inhibited , and an increase of free pool -catenin with its stabilization and translocation into the nucleus is developed [ 8 , 11 ] . in the nucleus -catenin acts as a transcriptional coactivator for the lef / tcf family of transcription factors . in the final step of the -catenin - lef / tcf signaling pathway , nuclear -catenin binds pontin52-tata - binding protein and displaces groucho - related gene or creb - binding protein corepressors from lef / tcf resulting in stimulation of transcription of important growth regulatory genes , including cyclin d1 and c - myc [ 8 , 13 ] ( figure 2 ) . there have been defined many other agents that work as physiological promoters of the wnt pathway , such as dishevelled - associated kinase ( dak ) , casein kinase i epsilon ( ck i ) , casein kinase ii ( ck ii ) , integrin - linked kinase ( ilk ) , b - cell lymphoma 9 ( bcl9 ) and bcl9 - 2/bcl9-like ( b9l ) , together with pygopus [ 8 , 10 , 1416 ] . these agents are involved in different parts of the wnt/-catenin pathway and lead to an increased accumulation of cellular -catenin with augmented assembly of wnt target genes . axin1 , axin2 , hmg - box transcriptional factor ( hbp1 ) , dapper1 ( dpr1 ) , chibby , and inhibitor of -catenin and tcf ( icat ) , on the contrary , represent complex negative regulators of the wntsignaling pathway . they antagonize the wnt pathway by decreasing the cellular -catenin , disrupting the -catenin / lef-1 complexes , and repressing the wnt target genes [ 7 , 13 , 1719 ] . there is strong evidence that defects in the wnt pathway are involved in the development of several types of solid tumors , for example , colorectal , prostate , and breast cancer [ 8 , 2022 ] . it has been known for a long time that hematological malignancies , such as chronic myeloid and lymphocytic leukemia , mantle cell lymphoma , multiple myeloma , and acute myeloid leukemia may occur partly because of the constitutive activation of wnt/-catenin canonical signaling pathway [ 23 , 24 ] . especially mutations in downstream components of the wnt pathway such as apc , axin , or -catenin have been found to be responsible for the genesis of human cancers due to aberrant signaling activity [ 9 , 25 ] . apc protein binds -catenin , retains it in the cytoplasm , and facilitates the proteolytic degradation of -catenin . abrogation of this negative regulation allows -catenin to translocate to the nucleus and to form a transcriptional activator complex with the dna - binding protein lef-1 . expression of mutant oncogenic forms of -catenin identified in cancer cells resulted in higher levels of transcriptional activity . the results suggest that a cancer pathway driven by wnt proteins , or mutant forms of -catenin , may involve the formation of a persistent transcriptionally active complex of -catenin and lef-1 . the treatment of the philadelphia chromosome - positive cml is highly effective due the inhibition of bcr - abl kinase activity by the tk inhibitor imatinib . nevertheless , it is difficult to achieve molecular remission , suggesting that leukemia stem cells ( lscs ) remain in the patient . in vivo after imatinib treatment , lscs not only remained but also accumulated increasingly in bone marrow of cml mice . in this context it is discussed that the wnt pathway plays a vital role in the survival and self - renewal of lsc . zhao et al . revealed that -catenin deletion causes a reduction in the ability of mice to develop bcr - abl - induced cml . intriguingly , genetic analysis of transformed blasts from patients in blast crisis has identified numerous members of the wnt/-catenin pathway as being activated . in addition to these data , evidence is emerging associating survival of blast cell with wnt activity , leading to the hope that wnt inhibitors will increase the likelihood of eradicating these cells . a comparison of the gene signatures of chronic , accelerated , and blast phases suggests that the progression of chronic phase to advanced phase cml ( accelerated phase and blast crisis ) is a two - step process , with new gene expression changes occurring early in accelerated phase before the accumulation of increased numbers of leukemia blast cells . in this process deregulation of the wnt/-catenin pathway seems to represent an important aspect . it has been demonstrated that the wnt signaling pathway is activated in cll cells and that uncontrolled wnt/-catenin signaling may contribute to the defect in apoptosis that characterizes this malignancy . it is thought to be responsible for the extended survival of cll cells in vivo . in primary cll cells , after upregulation of -catenin due to wnt stimulation , it cooperates with the transcription factor lef-1 , which is overly expressed in cll by more than three - thousand - fold compared to normal b cells . furthermore lef-1 is regarded as an essential regulator of pathophysiological , relevant genes in cll and several wnt/-catenin signaling components which fundamentally influence cll cell survival [ 14 , 33 ] . similar findings have been made by gutierrez et al . in analyzing the wnt pathway by using gene expression profiling . as a result aberrant regulation of wnt pathway target genes , ligands , and signaling members could be identified . here , especially the constitutive wnt pathway activation and the aberrant protein expression of lef-1 in cll was remarkable . even in patients with monoclonal b - cell lymphocytosis that is regarded as a premalignant condition of cll , an expression of lef-1 in cd19+/cd5 + cells could be identified . it has been revealed that certain wnt and wnt network target genes are expressed at higher or lower levels in cll compared with normal b cells . this includes upregulation of nuclear complex genes , as well as genes for cytoplasmatic proteins and wnt ligands and their related receptors . the balance between epigenetic downregulation of negative effector genes and increased expression of positive effector genes plays an outstanding role in the pathogenesis of cll . especially the epigenetic downregulation of wnt antagonists , such as dickkopf ( dkk ) and wif1 by hypermethylation , is one mechanism , perhaps the main mechanism that is permissive to active wnt signaling in cll [ 25 , 34 ] . furthermore , the overexpression of positive effectors in the wnt pathway is also involved in the pathogenesis of cll . in this model , dna methylation , histone modifications , and altered expression of microrna molecules interact to allow continuous wnt signaling . all these mechanisms result in a common consequence , the activation of lef-1/tcf transcription factors and subsequent target gene expression . a large number of wnt proteins such as wnt3 , wnt5b , wnt6 , and wnt10a , as well as the wnt receptor fzd3 , furthermore the wnt/-catenin - regulated transcription factor lef-1 and its downstream target cyclin d1 , are overexpressed in cll . were able to demonstrate that a pharmacological inhibitor of gsk3 , sb-216763 , activated -catenin - mediated transcription and enhanced the survival of cll lymphocytes . taken together , these results indicate that wnt signaling genes are overexpressed and active in cll . dickkopf1 ( dkk1 ) is known to antagonize wnt signaling by direct high - affinity binding to the extracellular domain of the wnt coreceptor lrp6 . in b cells from patients with cll , added dkk1 did not inactivate the wnt pathway . it is estimated that in cll cells every 6th lrp6 receptor is lacking the extracellular domain . normal b cells proved to have significantly higher levels of extracellular , dkk1-binding domain of lrp6 . on the other hand , a truncated lrp6 without extracellular dkk1-binding domain could lead to an uncontrollable activation of wnt signaling in cll . cancer stem cells ( cscs ) play a significant role in the development and recurrence of several cancers . at the same time while substantial progress has been made in developing therapeutics targeting in other signal pathways , the wnt pathway has remained an elusive therapeutic target . however , in the recent years specific inhibitors of this pathway have been keenly researched and developed . the variety of possible underlying mechanisms leading to -catenin / lef-1/tcf activation offers multiple options to target the aberrantly activated pathway in order to prevent target gene expression or their gene products to exert their tumorigenic function . over the last decade , numerous approaches have been developed to target the wnt/-catenin pathway in tumor cells , from antagonizing wnt ligand secretion or binding to promoting -catenin degradation to specifically blocking -catenin - mediated transcriptional activity . the binding of secreted wnt ligands to their receptors offers an attractive and accessible target for therapeutic regulation of these signaling pathways . wnt proteins , wnt receptors , and secreted wnt inhibitors are attractive as potential therapeutic agents and targets due to their extracellular location . wnt signaling results in a diverse array of downstream intracellular events , many of which are not fully understood . the targeting of this pathway at the most upstream site of pathway activation also provides a strategic advantage for therapy . there have been done promising researches and efforts on targeting the wnt pathway in solid tumors and hematology malignancies , for example , colorectal , breast , prostate cancer , multiple myeloma , and acute myeloid leukemia [ 21 , 22 , 3941 ] . based on current knowledge , there have not been made breakthrough advances targeting the wnt pathway in cml . however , the fact remains that -catenin in the wnt signaling pathway is essential for survival and self - renewal of lscs in cml , providing a new strategy for targeting these cells . particularly in patients with progress , relapse , or incompatibilities under the treatment with tk inhibitors further therapeutic developments have to be made . here , the wnt pathway could play a fundamental role . in contrast , recent researches indicate that the wnt pathway is an attractive candidate for developing targeted therapies for cll . as one of the first , lu et al . successfully targeted the wnt pathway in cll lymphocytes by using r - etodolac , an analog of a nonsteroidal anti - inflammatory drug , which diminished the wnt/-catenin signaling . furthermore lu et al . identified the diuretic agent ethacrynic acid ( ea ) as a wnt inhibitor . in vitro assays confirmed the inhibitory effect of ea on wnt/-catenin signaling . exposure of cll cells to ea decreased the expression of wnt/-catenin target genes , including lef-1 , cyclin d1 , and fibronectin . immune coprecipitation experiments revealed that ea could directly bind to lef-1 protein and destabilize the lef-1/-catenin complex . : a series of amides of ethacrynic acid were prepared and evaluated for their ability to inhibit wnt signaling and decrease the survival of cll cells . reduction of the alpha- , beta - unsaturated carbon - carbon double bond of ea abrogated both the inhibition of wnt signaling as well as the decrease in cll survival . preliminary mechanism of action studies suggests that these derivatives covalently modify sulfhydryl groups present on transcription factors important for wnt/-catenin signaling ( table 1 ) . another approach is the inhibition of the wnt pathway cascade further upwards by using salinomycin . salinomycin is an antibiotic potassium ionophore with the ability to inhibit the development of breast cancer stem cells . in wnt - transfected hek293 cells ingrain , another potassium ionophore with activity against cscs , revealed similar effects . in cll cells with constitutive wnt activation , nanomolar concentrations of salinomycin decreased the expression of wnt target genes such as lef-1 , cyclin d1 , and fibronectin , depressed lrp6 levels , and limited cell survival . these results indicate that ionic changes induced by salinomycin and related drugs inhibit proximal wnt signaling by interfering with lpr6 phosphorylation and thus impair the survival of cells that depend on wnt signaling at the plasma membrane . nitric oxide - donating acetylsalicylic acid ( no - asa ) the effect of the paraisomer of no - asa on cll cell survival in vitro and in a cll - like xenograft mouse model was analyzed by razavi et al . . apoptosis in primary cll cells was determined , and interference of no - asa with wnt/-catenin signaling was analyzed through immunoblots of different pathway members . cll - like jvm3 cells were subcutaneously inoculated into irradiated nude mice that were treated with 100 mg of para - no - asa / kg of body weight p.o . for 21 days . para - no - asa induced apoptosis in cll cells , whereas healthy blood cells were not affected . in addition , cleavage of -catenin and downregulation of -catenin / lef-1 targets were observed . para - no - asa exhibited strong antitumor efficacy in the xenograft mouse model with a tumor inhibition rate of 83.4% . para - no - asa selectively induces apoptosis in primary cll cells and efficiently reduces tumor growth in a cll - like xenograft model . as no - asa is orally available and is generally well tolerated , it might be a promising novel agent for cll therapy . used two small molecule inhibitors of the wnt/-catenin / lef-1 pathway ( cgp049090 and pkf115 - 584 ) in vitro and in vivo studies in order to antagonize lef-1 . by using nucleofection , small interfering rna- ( sirna- ) then the lc50 of the two small molecules was evaluated using atp - based cell viability assay . inhibition of lef-1 by sirna leads to increased apoptosis of cll cells and inhibited proliferation of jvm-3 cell lines ( subcutaneous xenograft model ) . the two small molecule inhibitors efficiently killed cll cells , whereas normal b cells were not significantly affected . in vivo studies exhibited tumor inhibition of 69% with cgp049090 and 57% with pkf115 - 584 . the wnt/-catenin signaling pathway plays an outstanding role in the development of chronic leukemia . a large number of the aberrant regulation mechanisms in the wnt pathway that lead to maintenance , progress , and relapse of chronic leukemia have been revealed . furthermore , in the recent years , there have been made promising experimental , preclinical researches , targeting the wnt pathway , particularly successfully in cll .

age - related decreases in muscle mass and strength are associated with decreased mobility , quality of life , and increased cardiovascular risk . coupled with the prevalence of obesity , the risk of death becomes substantially greater . resistance training ( rt ) has a well - documented beneficial impact on muscle mass and strength in young and older adults , although the high - intensity needed to elicit these adaptations may have a detrimental or negligible impact on vascular function , specifically on arterial stiffness . increased arterial stiffness is associated with systolic hypertension , left ventricular hypertrophy , and myocardial ischemia . therefore , improvements of muscle strength and arterial function are important in older adults . recently , whole - body vibration ( wbv ) exercise , a novel modality of strength training , has shown to exhibit similar results on muscle strength as rt in a wide - variety of populations , with the greatest impact in elderly individuals with limited muscle function . additionally , wbv training has been shown to have beneficial effects on vascular function by reducing arterial stiffness . this article reviews relevant publications reporting the effects of wbv on muscle strength and/or arterial stiffness . findings from current studies suggest the use of wbv training as an alternative modality to traditional rt to countermeasure the age - related detriments in muscle strength and arterial stiffness in older adults .

sarcopenia was originally defined as the age - related loss of appendicular lean ( muscle ) mass relative to height squared . recently , sarcopenia was redefined and either the loss of muscle strength and/or impaired physical performance was added to the loss of muscle mass.2 , 3 , 4 although reductions in muscle mass and strength are positively associated , the reduction in muscle strength exceeds that of mass . therefore , clark and manini proposed to define the loss of muscle strength as dynapenia . previous studies have shown that dynapenia combined with obesity is associated with increased cardiovascular risk.8 , 9 , 10 furthermore , obesity increases the risk of death in older adults with dynapenia . by contrast , high muscle strength reduces the mortality risk in individuals aged 5069 years . these findings suggest that maintaining muscle strength may attenuate , to some extent , the adverse cardiovascular risk associated with aging and obesity . another important age - related process is increased arterial stiffness [ pulse wave velocity ( pwv ) ] , which occurs primarily in the aorta . the age - related increase in pwv leads to isolated systolic hypertension due to impaired buffering function of the aorta . growing evidence suggests a strong negative relationship between increased pwv and reduced muscle mass , especially in the legs . abbatecola et al demonstrated that increased carotid - femoral pwv ( cfpwv ) , the gold - standard measure of aortic stiffness , was associated with limb muscle mass reduction . this negative relationship has been thoroughly investigated in asian populations using brachial - ankle pwv ( bapwv ) rather than cfpwv . bapwv is a composite of peripheral leg ( femoral - ankle pwv , fapwv ) and cfpwv , and thereby is a marker of systemic arterial stiffness . each increase of 1 m / s in bapwv is associated with an increase in cardiovascular event and mortality by 12% and 13% , respectively . a higher bapwv was found in women with greater sarcopenic class based on appendicular skeletal mass / height.2 , 16 more specifically , reduced thigh muscle mass was negatively associated with bapwv in nonobese middle - aged and older men . importantly , low muscle mass in obese adults , a condition termed sarcopenic obesity , has an additive adverse effect on bapwv.18 , 19 although little evidence exists on the inverse relationship between upper - body muscle strength and cfpwv in young healthy men , the association between handgrip or leg muscle strength and pwv is unknown in older adults . currently , the optimal exercise training for improving muscle mass / strength concurrently with arterial stiffness has not been determined . high - intensity rt has shown to be effective in increasing muscle mass and muscle strength in older adults.21 , 22 the american college of sports medicine recommends rt at 6080% of 1 repetition maximum ( 1rm ) for improving both muscle strength and mass ( quality ) in older adults.23 , 24 although high - intensity rt has shown to be effective for improving muscle quality , there are some concerns regarding a possible adverse effect on pwv.25 , 26 some studies have found that rt increases arterial stiffness in young adults.27 , 28 in a meta - analysis , the increase in pwv by 0.7 m / s after rt may not have important clinical adverse implications in young healthy adults . by contrast , other studies have demonstrated that pwv is not changed after rt in young and older men and women.21 , 29 , 30 , 31 , 32 apparently , upper - body exercises and high - intensity training would be factors involved in the potential increase of bapwv induced by rt.25 , 33 nevertheless , we reported that low - intensity rt , including only leg exercises , did not change pwv ( systemic , aortic , and leg ) in postmenopausal women . collectively , these data suggest that rt may not reduce pwv in older adults . exercise modalities targeting functional / structural improvements of the vascular and muscular systems are fundamental for the prevention and treatment of cardiovascular events . when integrating lifestyle modifications in order to attenuate the likelihood of these risk factors , individuals should not only consider their overall health benefit expectations , but also the practicality to adhere to the program ( e.g. , motivation , time - commitment , effort perception ) . during the past 1520 years , whole - body vibration ( wbv ) exercise has become an attractive strength training modality for many healthcare providers since its incorporation into clinical therapies and performance - based settings . additionally , the incorporation of a vibration - stimulus through passive or wbv in populations that are unable to perform intense exercise modalities may be a time - efficient alternative for reducing their risk of vascular dysfunction . while passive vibration ( pv ) propagates the vibration - induced oscillations to the exposed area without involving voluntary muscle contractions , wbv requires the individual to maintain / target respective joint angles while performing static or dynamic exercises over a vibrating platform . it has previously been shown that wbv promotes additional muscle contractions through the excitability of the spinal reflex via muscle spindles and -motor neurons . this may be a mechanism by which wbv exercise elicits an important effect on muscle strength . wbv training ( wbvt ) has shown to be an effective modality for the improvement of muscle strength in young and older adults ( table 1).35 , 36 , 37 , 38 some studies have reported that the increases in muscle strength after wbvt are comparable to those observed after conventional rt in healthy young males and females , as well as older men and postmenopausal women.35 , 37 , 39 , 40 , 41 , 44 , 45 , 46 one of the first studies demonstrated that 12 weeks of wbvt and rt induced comparable increases in isometric ( 16.6% ) and dynamic ( 9% ) knee extensor strength in young untrained lean females . these strength improvements were significant when compared to participants that performed the similar exercise protocol with no vibration or were in the nonexercising control group . roelants et al increased the duration of the study ( 24 weeks ) and found a greater increase in isometric knee extensor strength ( 24.4% ) in a similar population . in overweight and obese young women , we found that 6 weeks of wbvt induced a 6.5% increase in knee extensor strength . these findings were furthered by milanese et al when they increased the training duration to 10 weeks , the longer wbvt effectively increased knee extensor ( 14.2% ) , flexor ( 12.7% ) and press ( 15.8% ) strength . while the previous studies have noted similar results between wbvt and rt , they were conducted in young untrained lean adults . it is known that high - intensity exercise training decreases adherence , while lower intensity with low perceived effort is a successful exercise program in obese participants.46 , 53 obese postmenopausal women can perform 20 repetitions per set of a squat exercise , therefore the low - intensity explains the high adherence ( 98% ) to wbvt in this population . figueroa et al and milanese et al examined the effects of wbvt in young ( 21 years ) and middle - aged ( 47 years ) overweight and obese adult females . a significant increase in leg extension ( 6.5% ) was observed in the young overweight / obese population after 6 weeks of wbvt compared to the nonexercising controls . after 10 weeks of wbvt , milanese et al observed an increase in leg extension ( 14.2% ) , leg curl ( 12.7% ) and leg press ( 15.8% ) in middle - aged women . we and other groups have found that short - term ( 812 weeks ) wbvt increased leg muscle strength in overweight / obese postmenopausal women.36 , 46 , 49 , 54 , 55 due to the improvements on muscle strength after wbvt , several recent studies have been conducted in older adults to counteract the deleterious impact of aging , physical inactivity , and disease on muscle strength . in community - dwelling healthy older adults ( 6278 years ) , the increases in muscle strength after 848 weeks of wbvt were similar to those observed after rt ( 4.938.8% ) , but significantly greater when compared to sham and nonexercise controls.35 , 36 , 37 , 45 , 46 , 55 similarly , wbvt has increased muscle strength in patients with chronic stroke and diabetic neuropathy ( 22%).50 , 51 , 52 taken together , these findings suggest that the beneficial effect of wbvt on muscle strength gains are greater in populations with muscle weakness as a result of aging and/or diseases . while several studies have assessed the effects of pv and wbv exercise on muscle strength , bone mineral density , and cardiorespiratory function , only a limited number of studies have assessed the acute and chronic effects of this exercise modality on arterial stiffness . acute reductions on systemic arterial stiffness ( bapwv ) in young healthy males have been previously observed by otsuki et al and figueroa et al following 10 1-minute sets of static squats ( table 2 ) . otsuki et al initially evaluated the acute effects on bapwv and found significant decreases 20 minutes and 40 minutes following a single session of wbv , with values recovering to baseline 60 minutes after the last set . subsequently , figueroa et al examined the acute effects of the wbv protocol used by otsuki et al on aortic ( cfpwv ) , leg ( fapwv ) and bapwv . we observed significant reductions in fapwv within 30 minutes after the wbv protocol . yet , this was not detected for cfpwv and bapwv . furthermore , wong et al and koutnik et al examined the effects of acute pv applied to the posterior side of the legs ( ankles to glutes ) in healthy young men and post - stroke patients , respectively ; laying supine on the vibration platform . wong et al examined a session of 10 continuous minutes of pv and found decreases in bapwv and fapwv . koutnik et al utilized the same pv exposure to the legs and assessed fapwv and bapwv at 5 minutes , 15 minutes , and 30 minutes after the stimulus . pwvs were significantly decreased ( paretic and nonparetic sides ) 5 minutes after pv . after 15 minutes , the paretic and nonparetic fapwv remained significantly lower than baseline , yet only the nonparetic fapwv was different from control . practically , these previous findings suggest that acute exposure to either pv or exercise with wbv acutely decrease bapwv through local arterial effects independent of aortic stiffness . the effects of wbvt on pwv have been assessed following 6 weeks , 8 weeks , and 12 weeks in populations that exhibit heightened risks for developing cardiovascular diseases and physical disability ( e.g. , obesity , aging , hypertension ; table 2 ) . figueroa et al assigned young sedentary overweight / obese women to 6 weeks of wbvt three times a week . following wbvt , bapwv significantly decreased ( 0.9 0.3 m / s ) when compared to a nonexercising control period in a cross - over study . interestingly , this study combined static and dynamic semi - squats , wide - stand semi - squat , and calf - raise exercises over the vibrating platform . the dynamic exercises were performed with slow movements at a rate of 2 second concentric and 3 second eccentric phases , while the static movements were performed by maintaining the desired joint angle . moreover , figueroa et al48 , 54 examined the effects of 12 weeks of wbvt in postmenopausal women with pre- and stage 1-hypertension . participants underwent the same exercises , with the exception of the wide - stance ( they performed lunges instead ) . importantly , significant decreases in bapwv ( 1.23 m / s ) and fapwv ( 0.81 m / s ) were observed . moreover , lai et al investigated the effect of a 12-week wbvt program on bapwv in middle - aged and older adults . notably , participants in this study performed natural full - standing postures at a set frequency ( 30 hz ) , which were not utilized in any of the previously addressed studies . a recent study by figueroa et al evaluated the effects of combining wbvt with l - citrulline supplementation in postmenopausal women . in addition to the well - known reductions in bapwv and fapwv induced by wbvt alone , combining wbvt with a vasodilatory amino acid supplementation resulted in significant decreases in cfpwv ( 0.91 m / s ) , a reduction which had not been previously observed with low - intensity or high - intensity rt.32 , 33 wbv training ( wbvt ) has shown to be an effective modality for the improvement of muscle strength in young and older adults ( table 1).35 , 36 , 37 , 38 some studies have reported that the increases in muscle strength after wbvt are comparable to those observed after conventional rt in healthy young males and females , as well as older men and postmenopausal women.35 , 37 , 39 , 40 , 41 , 44 , 45 , 46 one of the first studies demonstrated that 12 weeks of wbvt and rt induced comparable increases in isometric ( 16.6% ) and dynamic ( 9% ) knee extensor strength in young untrained lean females . these strength improvements were significant when compared to participants that performed the similar exercise protocol with no vibration or were in the nonexercising control group . roelants et al increased the duration of the study ( 24 weeks ) and found a greater increase in isometric knee extensor strength ( 24.4% ) in a similar population . in overweight and obese young women , we found that 6 weeks of wbvt induced a 6.5% increase in knee extensor strength . these findings were furthered by milanese et al when they increased the training duration to 10 weeks , the longer wbvt effectively increased knee extensor ( 14.2% ) , flexor ( 12.7% ) and press ( 15.8% ) strength . while the previous studies have noted similar results between wbvt and rt , they were conducted in young untrained lean adults . it is known that high - intensity exercise training decreases adherence , while lower intensity with low perceived effort is a successful exercise program in obese participants.46 , 53 obese postmenopausal women can perform 20 repetitions per set of a squat exercise , therefore the low - intensity explains the high adherence ( 98% ) to wbvt in this population . figueroa et al and milanese et al examined the effects of wbvt in young ( 21 years ) and middle - aged ( 47 years ) overweight and obese adult females . a significant increase in leg extension ( 6.5% ) was observed in the young overweight / obese population after 6 weeks of wbvt compared to the nonexercising controls . after 10 weeks of wbvt , milanese et al observed an increase in leg extension ( 14.2% ) , leg curl ( 12.7% ) and leg press ( 15.8% ) in middle - aged women . we and other groups have found that short - term ( 812 weeks ) wbvt increased leg muscle strength in overweight / obese postmenopausal women.36 , 46 , 49 , 54 , 55 due to the improvements on muscle strength after wbvt , several recent studies have been conducted in older adults to counteract the deleterious impact of aging , physical inactivity , and disease on muscle strength . in community - dwelling healthy older adults ( 6278 years ) , the increases in muscle strength after 848 weeks of wbvt were similar to those observed after rt ( 4.938.8% ) , but significantly greater when compared to sham and nonexercise controls.35 , 36 , 37 , 45 , 46 , 55 similarly , wbvt has increased muscle strength in patients with chronic stroke and diabetic neuropathy ( 22%).50 , 51 , 52 taken together , these findings suggest that the beneficial effect of wbvt on muscle strength gains are greater in populations with muscle weakness as a result of aging and/or diseases . while several studies have assessed the effects of pv and wbv exercise on muscle strength , bone mineral density , and cardiorespiratory function , only a limited number of studies have assessed the acute and chronic effects of this exercise modality on arterial stiffness . acute reductions on systemic arterial stiffness ( bapwv ) in young healthy males have been previously observed by otsuki et al and figueroa et al following 10 1-minute sets of static squats ( table 2 ) . otsuki et al initially evaluated the acute effects on bapwv and found significant decreases 20 minutes and 40 minutes following a single session of wbv , with values recovering to baseline 60 minutes after the last set . subsequently , figueroa et al examined the acute effects of the wbv protocol used by otsuki et al on aortic ( cfpwv ) , leg ( fapwv ) and bapwv . we observed significant reductions in fapwv within 30 minutes after the wbv protocol . yet , this was not detected for cfpwv and bapwv . furthermore , wong et al and koutnik et al examined the effects of acute pv applied to the posterior side of the legs ( ankles to glutes ) in healthy young men and post - stroke patients , respectively ; laying supine on the vibration platform . wong et al examined a session of 10 continuous minutes of pv and found decreases in bapwv and fapwv . koutnik et al utilized the same pv exposure to the legs and assessed fapwv and bapwv at 5 minutes , 15 minutes , and 30 minutes after the stimulus . pwvs were significantly decreased ( paretic and nonparetic sides ) 5 minutes after pv . after 15 minutes , the paretic and nonparetic fapwv remained significantly lower than baseline , yet only the nonparetic fapwv was different from control . practically , these previous findings suggest that acute exposure to either pv or exercise with wbv acutely decrease bapwv through local arterial effects independent of aortic stiffness . the effects of wbvt on pwv have been assessed following 6 weeks , 8 weeks , and 12 weeks in populations that exhibit heightened risks for developing cardiovascular diseases and physical disability ( e.g. , obesity , aging , hypertension ; table 2 ) . figueroa et al assigned young sedentary overweight / obese women to 6 weeks of wbvt three times a week . following wbvt , bapwv significantly decreased ( 0.9 0.3 m / s ) when compared to a nonexercising control period in a cross - over study . interestingly , this study combined static and dynamic semi - squats , wide - stand semi - squat , and calf - raise exercises over the vibrating platform . the dynamic exercises were performed with slow movements at a rate of 2 second concentric and 3 second eccentric phases , while the static movements were performed by maintaining the desired joint angle . moreover , figueroa et al48 , 54 examined the effects of 12 weeks of wbvt in postmenopausal women with pre- and stage 1-hypertension . participants underwent the same exercises , with the exception of the wide - stance ( they performed lunges instead ) . importantly , significant decreases in bapwv ( 1.23 m / s ) and fapwv ( 0.81 m / s ) were observed . moreover , lai et al investigated the effect of a 12-week wbvt program on bapwv in middle - aged and older adults . notably , participants in this study performed natural full - standing postures at a set frequency ( 30 hz ) , which were not utilized in any of the previously addressed studies . a recent study by figueroa et al evaluated the effects of combining wbvt with l - citrulline supplementation in postmenopausal women . in addition to the well - known reductions in bapwv and fapwv induced by wbvt alone , combining wbvt with a vasodilatory amino acid supplementation resulted in significant decreases in cfpwv ( 0.91 m / s ) , a reduction which had not been previously observed with low - intensity or high - intensity rt.32 , 33 rt and wbvt improve muscle strength in older adults . however , a potential adverse effect of rt on pwv exists . by contrast , wbvt is associated with a decrease in systemic and leg arterial stiffness in young and older adults . further studies are needed to examine the long - term ( 6 months ) effects of wbvt on muscle mass and aortic pwv in individuals with high cardiovascular risk .

chronic subdural hematoma ( csdh ) is a common clinical entity treated by neurosurgeons with good cure rates . recurrence of the hematoma and failure of the brain to re - expand are commonly observed complications following surgery for csdh . an intracerebral or intraventricular hemorrhage following evacuation of csdh is very rarely observed , albeit with devastating consequences . we report the finding of a subependymal hemorrhage after evacuation of a csdh and discuss the probable causative mechanisms underlying this rare occurrence and the prevention strategies to avoid it .

chronic subdural hematoma ( csdh ) is a common clinical entity treated by neurosurgeons with very good cure rates . recurrence of the hematoma , infection , seizure , cerebral edema , tension pneumocephalus and failure of the brain to re - expand are well - recognized complications following surgery for csdh . an intracerebral or intraventricular hemorrhage following such an evacuation is very rarely observed ( incidence ~0.7 - 5% ) , albeit with devastating consequences . we report the finding of a subependymal hemorrhage after evacuation of a csdh and discuss the probable causative mechanisms underlying this rare occurrence and the prevention strategies to avoid it . past history revealed a minor fall with head injury 1 month ago for which no medical attention was sought . examination was unremarkable , except for the presence of pronator drift on the left side . ct ( computed tomography ) of the head showed a right csdh , measuring 15 mm in thickness and causing a 7 mm midline shift to the left [ figure 1a and b ] . after baseline investigations which documented a normal coagulation profile , the patient was taken for right frontal and parietal burr holes and drainage of the subdural hematoma under mac ( monitored anesthesia care ) with conscious sedation . after making the burr holes , the dura was coagulated with bipolar cautery and incised in a cruciate manner . the cut edges of the dura were further cauterized to shrink the dura and keep the dural opening patent . the subdural collection was allowed to drain without any negative pressure being applied in the subdural cavity . after the subdural hematoma had drained on its own accord ( owing to the pressure differential ) , the cavity was gently irrigated with normal saline till the returning fluid was clear . ct head done immediately post - surgery revealed a subependymal bleed in the right lateral ventricle , extending laterally into the thalamus and medially into the body of the lateral ventricle [ figure 1c and d ] . in spite of these findings in the post - operative ct , the patient did well in the post - operative period with complete resolution of headache and improvement of the subtle pyramidal deficit on the left side . ( a and b ) pre - operative ct head showing right chronic subdural hematoma . ( c and d ) post - operative ct head showing subependymal bleed in the right thalamus with extension into the lateral ventricle as mentioned above , do tend to jeopardize the good outcome normally expected in these patients ; but none are as devastating as the rare complication of intracerebral hemorrhage ( ich ) after evacuation of csdh . several causative mechanisms have been postulated to explain this complication following a simple surgical procedure . beneath the csdh , focal cerebral edema due to impeded venous drainage chronic dilatation of small arterial vessels and loss of carbon dioxide reactivity in the ischemic hemisphere could also contribute to the pathogenesis . possible pathogenic mechanisms , for ich in the thus compromised brain , include hemorrhage into previously undetected areas of contusion , damage to cerebral vasculature secondary to rapid peri - operative parenchymal shift , and sudden increase in cerebral blood flow ( post - decompression ) combined with focal disruption of autoregulation . compression by extra - axial collection decreases cerebral blood flow on the affected hemisphere and alters its vascular self - adjustment . some authors hypothesize that the rapid increase in cerebral blood flow ( after drainage ) in areas of the brain with altered vascular self - adjustment may be the most likely precipitating mechanism of intracerebral hemorrhage after surgical evacuation of csdhs . , as the compressed brain is more toward the surface , this mechanism is likely to explain superficial or lobar hematomas . we hypothesize that after sudden decompression of the brain , the differential expansile qualities of the solid ( brain ) and liquid ( csf ) components of the cranium may result in mechanical stress at the interface , which in turn , may cause rupture of the engorged subependymal veins ( due to prolonged compression ) . chronic sdh being a benign entity , neurosurgical outcome have to be optimized for this entity . considering that intracerebral hemorrhage inadvertently portends a poor outcome for the affected patients , preventive steps are imperative . these include gradual and graded decompression of the chronic sdh ( especially for large collections ) , use of closed drainage system which facilitates slow re - expansion of the brain , and careful control of blood pressure lability in the peri - operative period . the gradual and graded decompression can be achieved by covering the dural aperture with a cottonoid after draining around 15 to 20 ml of the subdural hematoma . after waiting for approximately 5 min , this procedure can be continued till the subdural pressures equalize with the atmospheric pressures , and at this juncture , saline washes can be instituted . this especially holds true for very large subdural hematomas , presenting with raised intra - cranial pressure . our patient was fortunate to have a good outcome as the bleed was moderate in size , however , we advocate taking steps to prevent such complications altogether .